Asgpr antibodies and uses thereof

ABSTRACT

The present invention generally relates to antibodies specific for asialoglycoprotein receptor (ASGPR) and their use for selectively delivering effector moieties that influence cellular activity. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies of the invention, and to methods of using them in the treatment of disease.

FIELD OF THE INVENTION

The present invention generally relates to antibodies specific forasialoglycoprotein receptor (ASGPR) and their use for selectivelydelivering effector moieties that influence cellular activity. Inaddition, the present invention relates to polynucleotides encoding suchantibodies, and vectors and host cells comprising such polynucleotides.The invention further relates to methods for producing the antibodies ofthe invention, and to methods of using them in the treatment of disease.

BACKGROUND

Asialoglycoprotein receptor (ASGPR) is a transmembrane receptor composedof two subunits, H1 and H2. The subunits are believed to oligomerizethroughout their extracellular stalk regions. ASGPR is a member of theC-type lectin family (calcium-ion dependent lectin) and mediates theendocytosis and degradation of a wide variety of desialylatedglycoproteins. ASGPR is selectively expressed on liver parenchymal cells(hepatocytes), which makes it an attractive target for liver-specifictherapies. Many liver diseases, e.g. hepatitis, liver cirrhosis orhepatocellular carcinoma (HCC), can be caused directly or indirectly byviral infection, such as hepatitis virus B (HBV) or C (HCV) infection.Chronic infection with HCV is one of the major causes of cirrhosis andHCC. Similarly, chronic HBV infection accounts for 5-10% of chronicliver disease and cirrhosis in the US. Approved therapies for HBV andHCV infection include interferons (IFN), such as interferon alpha.However, side effects have hampered development and widespread use ofthese therapies in many cases. Such IFN-associated side effects arethought to be due in part to induction of interferon-stimulated genes(ISGs) in peripheral blood cells following systemic exposure to IFN.Hence, to minimize side effects associated with IFN therapy for liverdiseases, and also to augment the antiviral effect of conventionalinterferons, it is desirable to selectively deliver IFN to the liver.ASGPR has been recognized as potential target molecule on hepatocytesfor such selective delivery. For example, WO 92/22310 describes anapproach for targeting interferon to the liver by conjugation ofrecombinant IFN to an asialoglycoprotein. In a similar approach, aninterferon molecule itself has been modified to produceasialo-interferon for binding to ASGPR (Eto and Takahashi, Nat Med 5,577-581 (1999)) More recently, an approach based on anti-ASGPR singlevariable domain (dAb) antibodies has been described (WO 2011/086143).

However, none of these approaches has been shown to be clinicallysuccessful so far, and there remains a need for improved targetingmolecules for selective delivery of therapeutic molecules, e.g.interferon, to the liver. The antibodies of the present inventioncombine several advantageous properties, which make them particularlysuitable for targeting effector moieties such as interferons toASGPR-expressing cells, e.g. for the treatment of liver diseases.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an antibody capable of specificbinding to asialoglycoprotein receptor (ASGPR), wherein the antibodycomprises a) the heavy chain variable region sequence of SEQ ID NO: 16and the light chain variable region sequence of SEQ ID NO: 14; b) theheavy chain variable region sequence of SEQ ID NO: 4 and the light chainvariable region sequence of SEQ ID NO: 2; c) the heavy chain variableregion sequence of SEQ ID NO: 8 and the light chain variable regionsequence of SEQ ID NO: 6; d) the heavy chain variable region sequence ofSEQ ID NO: 12 and the light chain variable region sequence of SEQ ID NO:10; e) the heavy chain variable region sequence of SEQ ID NO: 20 and thelight chain variable region sequence of SEQ ID NO: 18; f) the heavychain variable region sequence of SEQ ID NO: 24 and the light chainvariable region sequence of SEQ ID NO: 22; g) the heavy chain variableregion sequence of SEQ ID NO: 28 and the light chain variable regionsequence of SEQ ID NO: 26; h) the heavy chain variable region sequenceof SEQ ID NO: 4 and the light chain variable region sequence of SEQ IDNO: 30; i) the heavy chain variable region sequence of SEQ ID NO: 4 andthe light chain variable region sequence of SEQ ID NO: 32; j) the heavychain variable region sequence of SEQ ID NO: 4 and the light chainvariable region sequence of SEQ ID NO: 34; or k) the heavy chainvariable region sequence of SEQ ID NO: 24 and the light chain variableregion sequence of SEQ ID NO: 22.

In a particular embodiment, the antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 16 and the light chain variableregion sequence of SEQ ID NO: 14. In another particular embodiment, theantibody comprises the heavy chain variable region sequence of SEQ IDNO: 4 and the light chain variable region sequence of SEQ ID NO: 2.

In a further aspect, the invention provides an antibody capable ofspecific binding to ASGPR, wherein the antibody competes for binding toan epitope of ASGPR with an antibody comprising the heavy chain variableregion sequence of SEQ ID NO: 16 and the light chain variable regionsequence of SEQ ID NO: 14. In one embodiment, said antibody recognizesan epitope in the stalk region of ASGPR. In one embodiment, saidantibody is an affinity matured variant of the antibody comprising theheavy chain variable region sequence of SEQ ID NO: 16 and the lightchain variable region sequence of SEQ ID NO: 14. In one embodiment, saidantibody comprises a heavy chain variable region sequence that is atleast about 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:16, and a light chain variable region sequence that is at least about96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 14. In oneembodiment, said antibody comprises the light chain variable regionsequence of SEQ ID NO: 14 with one, two, three, four, five, six orseven, particularly two, three, four or five, amino acid substitutions.In one embodiment, said antibody comprises the heavy chain variableregion sequence of SEQ ID NO: 16 with one, two, three, four, five, sixor seven, particularly two, three, four or five, amino acidsubstitutions.

In still a further aspect, the invention provides a an antibody capableof specific binding to ASGPR, wherein the antibody competes for bindingto an epitope of ASGPR with an antibody comprising the heavy chainvariable region sequence of SEQ ID NO: 4 and the light chain variableregion sequence of SEQ ID NO: 2. In one embodiment, said antibodyrecognizes an epitope in the carbohydrate recognition domain (CRD) ofASGPR. In one embodiment, said antibody is an affinity matured variantof the antibody comprising the heavy chain variable region sequence ofSEQ ID NO: 4 and the light chain variable region sequence of SEQ ID NO:2. In one embodiment, said antibody comprises a heavy chain variableregion sequence that is at least about 96%, 97%, 98% or 99% identical tothe sequence of SEQ ID NO: 4, and a light chain variable region sequencethat is at least about 96%, 97%, 98% or 99% identical to the sequence ofSEQ ID NO: 2. In one embodiment, said antibody comprises the light chainvariable region sequence of SEQ ID NO: 2 with one, two, three, four,five, six or seven, particularly two, three, four or five, amino acidsubstitutions. In one embodiment, said antibody comprises the heavychain variable region sequence of SEQ ID NO: 4 with one, two, three,four, five, six or seven, particularly two, three, four or five, aminoacid substitutions. In one embodiment, said antibody comprises a) theheavy chain variable region sequence of SEQ ID NO: 4 and the light chainvariable region sequence of SEQ ID NO: 36; b) the heavy chain variableregion sequence of SEQ ID NO: 4 and the light chain variable regionsequence of SEQ ID NO: 38; c) the heavy chain variable region sequenceof SEQ ID NO: 8 and the light chain variable region sequence of SEQ IDNO: 40; d) the heavy chain variable region sequence of SEQ ID NO: 12 andthe light chain variable region sequence of SEQ ID NO: 42; e) the heavychain variable region sequence of SEQ ID NO: 20 and the light chainvariable region sequence of SEQ ID NO: 44; f) the heavy chain variableregion sequence of SEQ ID NO: 24 and the light chain variable regionsequence of SEQ ID NO: 46; or g) the heavy chain variable regionsequence of SEQ ID NO: 28 and the light chain variable region sequenceof SEQ ID NO: 48.

In one embodiment, the antibody of the invention is capable of specificbinding to human and cynomolgus monkey ASGPR. In one embodiment, theantibody binds to human ASGPR with an dissociation constant (K_(D)) ofsmaller than 1 μM, particularly smaller than 100 nM, more particularlysmaller than 1 nM, when measured as Fab fragment by Surface PlasmonResonance (SPR). In one embodiment, the antibody binds to human ASGPRwith a K_(D) of smaller than 1 μM, particularly smaller than 500 nM,more particularly smaller than 100 nM or even smaller than 10 nM, whenmeasured as IgG₁ by fluorescence resonance energy transfer (FRET). Inone embodiment, the antibody does not compete with a natural ligand ofASGPR for binding to ASGPR. In a specific embodiment, said naturalligand of ASGPR is asialofetuin. In one embodiment, the antibody doesnot detectably bind to CLEC10A, particularly human CLEC10A. In oneembodiment, the antibody does not specifically bind to cells lackingASGPR expression, particularly human cells, more particularly humanblood cells. In one embodiment, the antibody is internalized into a cellexpressing ASGPR upon binding of the antibody to ASGPR on the surface ofsaid cell. In a specific embodiment, the antibody is recycled back tothe surface of said cell at about the same rate as it is internalizedinto said cell. In one embodiment, the antibody does not significantlyinduce downregulation of ASGPR expression at the surface of a cell uponbinding of the antibody to ASGPR on the surface of said cell.

In one embodiment, the antibody of the invention is a human antibody. Inone embodiment, the antibody comprises a human Fc region, particularlyan IgG Fc region, more particularly an IgG₁ Fc region. In oneembodiment, the antibody is a full-length antibody. In one embodiment,the antibody is an IgG class antibody, particularly an IgG₁ subclassantibody. In one embodiment, the antibody comprises in the Fc region amodification reducing binding affinity of the antibody to an Fcreceptor, particularly an Fcγ receptor. In a specific embodiment, saidFc receptor is an activating Fc receptor. In a further specificembodiment, said Fc receptor is selected from the group of FcγRIIIa(CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). In a morespecific embodiment, said Fc receptor is FcγRIIIa, particularly humanFcγRIIIa. In one embodiment, the antibody comprises an amino acidsubstitution in the Fc region at a position selected from P329, L234 andL235 (EU numbering). In one embodiment, the antibody comprises the aminoacid substitutions P329G, L234A and L235A in the Fc region (EUnumbering). In a further embodiment, the antibody comprises in the Fcregion a modification promoting heterodimerization of two non-identicalantibody heavy chains. In a specific embodiment, said modification is aknob-into-hole modification, comprising a knob modification in one ofthe antibody heavy chains and a hole modification in the other one ofthe two antibody heavy chains. In one embodiment, the antibody comprisesa modification within the interface between the two antibody heavychains in the CH3 domain, wherein i) in the CH3 domain of one heavychain, an amino acid residue is replaced with an amino acid residuehaving a larger side chain volume, thereby generating a protuberance(“knob”) within the interface in the CH3 domain of one heavy chain whichis positionable in a cavity (“hole”) within the interface in the CH3domain of the other heavy chain, and ii) in the CH3 domain of the otherheavy chain, an amino acid residue is replaced with an amino acidresidue having a smaller side chain volume, thereby generating a cavity(“hole”) within the interface in the second CH3 domain within which aprotuberance (“knob”) within the interface in the first CH3 domain ispositionable. In one embodiment, the antibody comprises the amino acidsubstitution T366W and optionally the amino acid substitution S354C inone of the antibody heavy chains, and the amino acid substitutionsT366S, L368A, Y407V and optionally Y349C in the other one of theantibody heavy chains.

In one aspect, the invention provides an antibody capable of specificbinding to ASGPR according to any of the above embodiments, wherein aneffector moiety is attached to the antibody. In one embodiment, not morethan one effector moiety is attached to the antibody. In one embodimentsaid effector moiety is a cytokine molecule. In one embodiment, saidcytokine molecule is fused at its amino-terminal amino acid to thecarboxy-terminal amino acid of one of the antibody heavy chains,optionally through a peptide linker. In one embodiment, said cytokinemolecule is a human cytokine. In one embodiment, said cytokine moleculeis an interferon molecule. In a specific embodiment, said interferonmolecule is interferon alpha, particularly human interferon alpha, moreparticularly human interferon alpha 2 (see SEQ ID NO: 138) or humaninterferon alpha 2a (see SEQ ID NO: 139). In one embodiment, wherein thecytokine molecule is an interferon molecule, the antibody has anti-viralactivity in cells expressing ASGPR on their surface. In a specificembodiment, the antibody has no anti-viral activity in cells notexpressing significant levels of ASGPR on their surface. In a furtherspecific embodiment, said anti-viral activity is selected frominhibition of viral infection, inhibition of virus replication,inhibition of cell killing and induction of interferon-stimulated genes.

The invention further provides a polynucleotide encoding the antibody ofthe invention. Further provided is a vector, particularly an expressionvector, comprising the polynucleotide of the invention. In anotheraspect, the invention provides a host cell comprising the polynucleotideor the vector of the invention. The invention also provides a method forproducing an antibody of the invention, comprising the steps of (i)culturing the host cell of the invention under conditions suitable forexpression of said antibody, and (ii) recovering said antibody. Alsoprovided is an antibody capable of specific binding to ASGPR, producedby said method.

In one aspect, the invention provides a pharmaceutical compositioncomprising the antibody of the invention and a pharmaceuticallyacceptable carrier. The antibody or the pharmaceutical composition ofthe invention is also provided for use as a medicament, and for use inthe treatment or prophylaxis of a liver disease, specifically a viralinfection, more specifically hepatitis virus infection, particularlyhepatitis B virus (HBV) infection. The antibody or the pharmaceuticalcomposition of the invention is also provided for use in the treatmentor prophylaxis of cancer, specifically liver cancer, more specificallyhepatocellular carcinoma (HCC). Further provided is the use of theantibody of the invention for the manufacture of a medicament for thetreatment of a disease in an individual in need thereof, and a method oftreating a disease in an individual, comprising administering to saidindividual a therapeutically effective amount of a compositioncomprising the antibody of the invention in a pharmaceuticallyacceptable form. In one embodiment, said disease is a liver disease. Ina more specific embodiment, said liver disease is a viral infection. Inan even more specific embodiment, said liver disease is hepatitis virusinfection, particularly HBV infection. In another embodiment, saiddisease is cancer. In a more specific embodiment, said cancer is livercancer. In an even more specific embodiment, said liver cancer ishepatocellular carcinoma (HCC). In one embodiment, said individual is amammal, particularly a human. In a further aspect, the antibody of theinvention is provided for targeting a cell expressing ASGPR in anindividual. Also provided is a method for targeting a cell expressingASGPR in an individual, comprising administering to said individual acomposition comprising the antibody of the invention in apharmaceutically acceptable form. In one embodiment, said cell is aliver cell, particularly a hepatocyte. In one embodiment, saidindividual is a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the generated antigen constructs. Thenucleotide sequences of all antigens were fused to the C-terminal end ofa human-derived IgG₁ Fc sequence. The ASGPR1- and CLEC10A-derived CRDswere fused to a sequence encoding an Fc(hole) fragment and co-expressedwith a sequence encoding an Fc(knob) fragment resulting in a monomericdisplay per Fc dimer. From left to right: Fc-CRD (ASGPR1), Fc-stalk(ASGPR1), Fc-stalk-CRD (ASGPR1), Fc-CRD (CLEC10A), Fc-stalk (CLEC10A),Fc-stalk-CRD (CLEC10A). Thick, curved line: (G₄S)₃ linker; thick,straight line: Xa and IgAse cleavage site.

FIG. 2. Schematic overview of the generation of the generic Fab libraryrandomized in the CDR3 regions of the heavy and the light chain. In afirst step, three PCR fragments were generated which were then fused by(splicing by overlapping extension; SOE) PCR. The final fragment wasgel-purified, digested with NcoI/NheI alongside with similarly treatedacceptor phagemid, ligated and transformed into bacteria. PCR1 (A, B,C): (1) LMB3; (2) (A) VI_(—)3_(—)19_L3r_V/(B) VI_(—)3_(—)19L3r_HV/(C)VI_(—)3_(—)19L3r_HLV. PCR2: (3) RJH80; (4) DP47CDR3_ba (mod). PCR3 (A,B, C): (5) (A) DP47 v4 4/(B) DP47 v4 6/(C) DP47 v4 8; (6) fdseqlong.

FIG. 3. Binding analysis of selected anti-human ASGPR H1-specific clonesto HepG2 cells as human IgG₁ antibodies. Antibody concentration was 30μg/ml. An isotype control antibody served as a negative control.

FIG. 4. FRET analysis on transiently transfected cells expressing atransmembrane ASGPR H1-SNAP tag fusion protein labeled with terbium.Analysis was done by adding antibodies at a concentration ranging from50-0.39 nM followed by addition of an anti-humanFc-d2 (final 200 nM perwell) as acceptor molecule. Specific FRET signal was measured after 3 hand KD values were calculated (KD_(51A12)=200 nM, KD_(R7F12)=22 nM,KD_(R9E10)=6.2 nM, KD_(R5C2)=5.9 nM, KD_(4F3)=4.5 nM).

FIGS. 5 and 6. Competition of asialofetuin, a natural ligand for ASGPR,and anti-ASGPR H1 antibodies. HepG2 cells were pre-incubated withlabeled asialofetuin before indicated antibodies were added in adilution row to the cells. Binding of both components to the cells wasanalyzed by FACS analysis. (A) antibody detection; (B) asialofetuindetection.

FIG. 7. Internalization analysis of the two anti-human ASGPR H1 antibodyclones 51A12 and 4F3 as IgGs. (A) antibodies were incubated with HepG2cells at 4° C. to prevent internalization, and washed at 4° C. beforethe cells were cultured in pre-warmed medium and incubated at 37° C. forup to 120 min. Samples were taken at indicated time points, labeled withthe secondary antibody on ice and fixed using PFA. (B) Same steps wereperformed as described under (A) but antibodies were incubated with thecells at 37° C. allowing ASGPR to internalize. (C) Same steps wereperformed as described under (A), but using directly FITC-labeledantibodies. Cell surface-bound antibodies were detected usingPE-conjugated anti-Fc antibody. (D) Same experiment as under (C) butshowing FITC signal, representing both surface-exposed and internalizedantibodies.

FIG. 8. Randomization strategy of the LCDR3 region of clone 51A12. Shownare (A) the LCDR3 protein sequence of the parental clone 51A12, (B) theLCDR3 protein sequence of the plasmid serving as a template for thelibrary without cysteines and glycosylation sequence, and (C) therandomized positions in LCDR3. During generation of the library,trinucleotide primers allow to exclude triplets encoding cysteines oramino acids contributing to the formation of a glycosylation site.

FIG. 9. Schematic overview of the generation of the affinity maturationlibrary randomized in LCDR3 of the template 51A12 (A82G, C112S, C113S,S116A) (SEQ ID NO: 33). In a first step, two PCR fragments weregenerated which were then fused by (SOE) PCR. The final fragment wasgel-purified, digested with NcoI/PstI alongside with similarly treatedacceptor phagemid, ligated and transformed into bacteria. PCR1: (1)LMB3, (2) LCDR3rev. PCR2: (3) LCDR3rand, (4) fdseqlong.

FIG. 10. Binding analysis of affinity-matured 51A12-derived clones toHepG2 cells as human Fab fragments. Fab concentrations of 10, 3.3, and1.1 μg/ml were used. The parental clone 51A12 (SEQ ID NOs 2 and 4),51A12 (S116A) (SEQ ID NOs 4 and 30), a clone devoid of the glycosylationsequence, and 51A12 (A82G, C112S, C113S, S116A) (SEQ ID NOs 4 and 34),the template clone for the affinity maturation library, served ascontrols.

FIG. 11. Binding analysis of affinity-matured 51A12-derived clones toHepG2 cells as human IgG₁ antibodies. Concentrations in a dilution rowranging from 0.01 to 20 μg/ml were used. The parental clone 51A12 (SEQID NOs 2 and 4) served as a control (A). Binding analysis to Hela cellsat a concentration of 10 μg/ml was used as a negative control (B).

FIG. 12. Schematic diagram of the generated antibody-cytokineconjugates. The gene encoding interferon-α2a was fused to the C terminalend of an ASGPR H1-specific antibody heavy chain comprising a knobmodification. While bivalent ASGPR binding of the antibody-cytokineprotein was achieved by co-expression of the corresponding ASGPRH1-specific heavy chain comprising a hole modification and the lightchain ((A), 2:1 valency), expression of a Fc(hole) fragment sequenceresulted in a monomeric antibody-cytokine conjugate with only one ASGPRH1-specific binding site per molecule ((B), 1:1 valency). Small blackdots: modification preventing FcγR binding (for example L234A L235AP329G). Large black dots: modification promoting heterodimerization (forexample knob-into-hole).

FIGS. 13-19. Purification and analytical characterization of selectedantibody-IFNα immunoconjugates (FIG. 13: 51A12 kih IgG-IFNα; FIG. 14:4F3 kih IgG-IFNα; FIG. 15: 51A12 (C7) kih IgG-IFNα; FIG. 16: 51A12 (C1)kih IgG-IFNα; FIG. 17: 51A12 (E7) kih IgG-IFNα; FIG. 18: isotype controlkih IgG-IFNα; FIG. 19: monovalent 51A12 kih IgG-IFNα). The purificationmethod involved an affinity step (protein A) (A) followed by sizeexclusion chromatography (Superdex 200, GE Healthcare) (B). The finalproduct was analyzed and characterized by analytical size exclusionchromatography (Superdex 200 column) (C) and microfluidic proteinanalysis (Caliper) or SDS-PAGE (D).

FIG. 20. Binding selectivity of ASGPR-specific IgG kih IFNα fusionconstructs 51A12 (A) and 4F3 (B). HepG2, primary human hepatocytes,Huh-7 cells, A549 cells, Hela cells, and 293T cells were incubated with1 μg/ml 51A12-IFNα (A) or 4F3-IFNα (B) for 45 min on ice. After threewashes, cells were stained with secondary goat anti-human IgG antibodyon ice for 30 min, and the cells were washed three times before beinganalyzed using a Calibur flow cytometer. Binding to human PBMC wasperformed by using 1 μg/ml of directly labeled 51A12 IgG kih IFNα (A)and 4F3 IgG kih IFNα (B) using the Zenon® R-Phycoerythrin Human IgGLabeling Kit according to manufacturer's instructions, isotype IgG kihIFNα and a CD81 mAb were used as negative and positive controls,respectively.

FIG. 21. Binding saturation curves of ASGPR mAb 4F3-IFNα on primaryhuman hepatocytes and HepG2 cells. Binding saturation of ASGPR mAb4F3-IFNα on human hepatocytes derived from three different donors (A-C)and HepG2 cells (D). Cells were incubated with serially diluted 4F3 IgGkih IFNα for 45 min on ice. After three washes, cells were stained withsecondary goat anti-human IgG antibody on ice for 30 min, and washedagain three times before being analyzed using a Calibur flow cytometer.

FIG. 22. Analysis of the surface-exposed levels of ASGPR over time inpresence of specific antibodies. HepG2 cells were incubated with eitherASGPR-specific clone 51A12 IgG or the corresponding monovalent orbivalent antibody-cytokine conjugate. Cell samples were taken after upto 5 hrs and binding of the IgG constructs to ASGPR was measured bydetection of either the antibody (A) or the cytokine (B). An anti-CD20antibody (GA101) was used as a negative control.

FIG. 23. Rapid internalization of clone 51A12 antibody-cytokineconjugate. Alexa488-labeled 51A12 IgG kih IFNα construct was incubatedwith HepG2 cells and ASGPR-mediated internalization of the construct wasrecorded by confocal microscopy for 1 h over 10 stacks (z-level).Binding of the antibody-cytokine conjugate on the cell surface occurs inclusters rather than homogenous distribution (A). Once bound to the cellsurface, the conjugate internalizes very rapidly in vesicles which aretransported into the cell body (B, single cell surrounded). Vesicles arethen recycled back to the apical side of the cell (not shown).

FIG. 24. Antiviral activity of ASGPR mAb-IFNα molecules and othercontrol IFN molecules in EMCV CPE (A) and HCV replicon (B) assays. (A)Hela cells were pretreated with serially diluted IFN molecules for 3 hbefore adding EMCV virus. Cells were cultured for 24 h, and cellviability was measured by adding CellTiter Glo. (B) Huh-7 2209 repliconcells were treated with serially diluted IFN molecules and luciferaseactivity was measured after 3 days.

FIG. 25. ISG induction by 51A12-IFNα in various hepatic and non-hepaticcells. Hepatic cells (primary hepatocytes (B) and HepG2 (A)) andnon-hepatic cells (human PBMC (D) and Hela (C)) were treated withvarious serially diluted IFNα molecules for 6 h, total RNA was extractedand TaqMan RT-PCR was used to quantify ISG MX1 (A, C) and Rsad2 (B, D)gene expression. Data shown are from three or more experiments.

FIG. 26. Sustained ISG induction by 51A12-IFNα and 4F3-IFNα in primaryhuman hepatocytes (PHH) (B) and Huh7 (A) cells. Primary humanhepatocytes (PHH) and Huh7 cells were treated with serially diluted IFNαmolecules for 6 h (left) and 72 h (right), total RNA was extracted andTaqMan RT-PCR was used to quantify ISG MX1 (A) and Rsad2 (B) geneexpression.

FIG. 27. Representative ISG expression in monkey liver samples. Monkeyliver samples from the four dose groups collected at different timepoints were analyzed by microarray. Expression of four representativeISG genes is shown in a 3D graph. The four dose groups are indicated.For each dose group, from left to right, bars represent ISG foldinduction at day −5, day 2, day 4, and day 8 of the 3 monkeys.

FIG. 28. Blood and liver gene expression heatmaps (IFN module M3.1).mRNA microarray analysis was performed on blood PBMC and liver biopsysamples, and their IFNα response was analyzed with gene modulesdetermined from blood transcriptomics studies (Chaussabel et al. (2008),Immunity 29, 150-64). In panel (A), the fold-change expression valuesfrom baseline (see inset) for the genes of the interferon module M3.1were plotted in heatmap form for both blood and liver samples using theR statistics package (www.r-project.org). Non-supervised hierarchicalclustering of the liver interferon-induced genes reveals a highlyinduced subset (dashed rectangle) in the 10 μg/kg dose of 51A12 but notthe isotype-IFNα compound at days 1 and 3. (B) This subset of genes wasplotted for both liver and blood. Non-supervised hierarchical clusteringthis subset reveals a differential pattern of expression between bloodand liver where the upper half of the heatmap shows induction in liverfor 51A12 but not isotype-IFNα at the 10 μg/kg dose and in the lowerhalf, induction in blood only for isotype-IFNα at the high dose.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following. “Asialoglycoprotein receptor”, abbreviated asASGPR, refers to any native ASGPR from any vertebrate source, includingmammals such as primates (e.g. humans), non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed ASGPR as wellas any form of ASGPR that results from processing in the cell. The termalso encompasses naturally occurring variants of ASGPR, e.g., splicevariants or allelic variants. In one embodiment, the antibody of theinvention is capable of specific binding to human ASGPR, particularlyhuman ASGPR H1, more particularly the extracellular domain of humanASGPR H1.

The amino acid sequence of human ASGPR H1 (also known as CLEC4H1) isshown in UniProt (www.uniprot.org) accession no. P07306 (version 131),or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_(—)001662.1. The extracellulardomain (ECD) of human ASGPR H1 extends from amino acid position 62 to291. The nucleotide and amino acid sequences of a human ASGPR H1 ECDfused to a human Fc region is shown in SEQ ID NOs 129 and 130,respectively. The ASGPR H1 ECD comprises the stalk region, which extendsfrom amino acid position 62 of the full sequence to around amino acidposition 160 (SEQ ID NOs 123 and 124 show nucleotide and amino acidsequences of a human ASGPR H1 stalk region fused to a human Fc region),and the carbohydrate recognition domain (CRD), which extends from aroundamino acid position 161 of the full sequence to around amino acidposition 278 (SEQ ID NOs 117 and 118 show nucleotide and amino acidsequences of a human ASGPR H1 CRD region fused to a human Fc region). Inone embodiment, the antibody is also capable of binding to cynomolgusASGPR, particularly cynomolgus ASGPR H1, more particularly theextracellular domain of cynomolgus ASGPR H1. The sequence of cynomolgusASGPR H1 is shown in NCBI GenBank accession no. EHH57654.1. SEQ ID NOs131 and 132 show the nucleotide and amino acid sequences, respectively,of a cynomolgus ASGPR H1 ECD fused to a human Fc region.

By “human CLEC10A” is meant the protein described in UniProt accessionno. Q8IUN9 (version 86), particularly the extracellular domain of saidprotein which extends from amino acid position 61 to amino acid position316 of the full sequence. SEQ ID NOs 133 and 134 show the nucleotide andamino acid sequences, respectively, of a human CLEC10A ECD fused to ahuman Fc region.

As used herein, the term “conjugate” refers to a fusion polypeptidemolecule that includes one effector moiety and a further peptidemolecule, particularly an antibody. A fusion protein of an antibody andan effector moiety is referred to as an “immunoconjugate”. An(immune)conjugate as referred to herein is a fusion protein, i.e. thecomponents of the (immune)conjugate are linked to each other bypeptide-bonds, either directly or through peptide linkers.

An “epitope” is a region of an antigen that is bound by an antibody. Theterm refers to a site (e.g. a contiguous stretch of amino acids or aconformational configuration made up of different regions ofnon-contiguous amino acids) on a polypeptide macromolecule to which anantibody binds, forming an antibody-antigen complex.

An “antibody that competes for binding to an epitope” with a referenceantibody refers to an antibody that blocks binding of the referenceantibody to its antigen in a competition assay by 50% or more, andconversely, the reference antibody blocks binding of the antibody to itsantigen in a competition assay by 50% or more. An exemplary competitionassay is provided herein.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antibody to bind to a specific antigencan be measured either through an enzyme-linked immunosorbent assay(ELISA) or other techniques familiar to one of skill in the art, e.g.Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcoreinstrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), andtraditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Inone embodiment, the extent of binding of an antibody to an unrelatedprotein is less than about 10% of the binding of the antibody to theantigen as measured, e.g. by SPR. In certain embodiments, an antibodythat binds to the antigen has a dissociation constant (K_(D)) of ≦1 μM,≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸M orless, e.g. from 10⁻⁸M to 10⁻¹³ M, e.g. from 10⁻⁹M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (K_(D)), which is the ratio of dissociation andassociation rate constants (k_(off) and k_(on), respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

By “internalization” is meant the removal of a molecule from the surfacefrom a cell by uptake of said molecule into the intracellular space. Aparticular form of internalization is receptor-mediated endocytosis,which occurs upon binding of a ligand or antibody to a cell surface(membrane-spanning) receptor, by inward budding of plasma membranevesicles containing the receptor and bound ligand or antibody.Internalization can be assessed using art-known techniques. A methodbased on determination of protein levels on the cell surface by FACS isdescribed in the Examples hereinbelow.

The term “recycling” as used herein refers to the re-appearance of amolecule on the surface of a cell after previous internalization of saidmolecule into said cell. Recycling implies that the molecule is notdegraded within the cell upon internalization. If recycling occurs atthe same rate as internalization, a dynamic steady state is reachedwherein the number of molecules on the cell surface stays essentiallyconstant. Recycling can be detected by techniques well known in the art,e.g. by determination of protein levels on the cell surface by FACS orusing (confocal) microscopy methods as described in the Exampleshereinbelow.

By “downregulation” is meant the reduction of the copy number of acertain protein, e.g. a cell surface receptor, within or at the surfaceof a cell. Downregulation as used herein particularly refers to areduction in the copy number of a cell surface protein present at thecell surface, e.g. by internalization and/or degradation, or reducedexpression. Downregulation of protein levels can be detected by variousmethods established in the art, including e.g. Western blot (for overallprotein levels) or FACS (for surface protein levels).

As used herein, the term “effector moiety” refers to a molecule,particularly a polypeptide molecule (e.g. a protein or glycoprotein),that influences cellular activity, for example, through signaltransduction or other cellular pathways. Accordingly, the effectormoiety can be associated with receptor-mediated signaling that transmitsa signal from outside the cell membrane to modulate a response in a cellbearing one or more receptors for the effector moiety. In oneembodiment, an effector moiety can elicit a cytotoxic response in cellsbearing one or more receptors for the effector moiety. In anotherembodiment, an effector moiety can elicit a proliferative response incells bearing one or more receptors for the effector moiety. In anotherembodiment, an effector moiety can elicit differentiation in cellsbearing receptors for the effector moiety. In another embodiment, aneffector moiety can alter expression (i.e. upregulate or downregulate)of an endogenous cellular protein in cells bearing receptors for theeffector moiety. Non-limiting examples of effector moieties includesmall molecules, cytokines, growth factors, hormones, enzymes,substrates, and cofactors. The effector moiety can be associated withthe antibody in a variety of configurations.

The term “attached” includes linkage by any kind of interaction,including chemical or peptide bonds.

“Fused” refers to components that are linked by peptide bonds, eitherdirectly or via one or more peptide linkers.

As used herein, the term “cytokine” refers to a molecule that mediatesand/or regulates a biological or cellular function or process (e.g.immunity, inflammation, and hematopoiesis). The term “cytokine” as usedherein includes lymphokines, chemokines, monokines, and interleukins.Examples of cytokines include, but are not limited to, GM-CSF, IL-1α,IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-α,IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. Particularcytokines are interferons (IFN), particularly IFN-α. In particularembodiments the cytokine is a human cytokine. The sequences ofparticular cytokines, human IFNα2 and IFNα2a, are shown in SEQ ID NOs138 and 139, respectively.

“Interferon-stimulated genes” (ISGs) refers to genes the expression ofwhich in a cell can be stimulated by contacting said cell with aninterferon molecule, particularly an IFNα molecule. Typically, ISGscomprise a recognition sequence (e.g. an interferon-stimulated responseelement (ISRE)) to which one or more interferon-activated signalingmolecules (e.g. STATs) can bind, thereby leading to enhanced expressionof the ISG. Examples of ISGs include MX1 (myxovirus restistance 1, alsoknown as interferon-induced protein p78), RSAD2 (radical S-adenosylmethionine domain containing 2, also known as cytomegalovirus-inducedgene 5), HRASLS2 (HRAS-like suppressor 2), IFIT1 (interferon-inducedprotein with tetratricopeptide repeats 1), and IFITM2(interferone-induced transmembrane protein 2).

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In one embodiment,the effector moiety is a single-chain peptide molecule. Non-limitingexamples of single-chain effector moieties include cytokines, growthfactors, hormones, enzymes, substrates, and cofactors. When the effectormoiety is a cytokine and the cytokine of interest is normally found as amultimer in nature, each subunit of the multimeric cytokine issequentially encoded by the single-chain of the effector moiety.Accordingly, non-limiting examples of useful single-chain effectormoieties include GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-10, IL-12, IL-15, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β,TGF-β, TNF-α, and TNF-β.

As used herein, the term “effector moiety receptor” refers to apolypeptide molecule capable of binding specifically to an effectormoiety. Where an effector moiety specifically binds to more than onereceptor, all receptors that specifically bind to the effector moietyare “effector moiety receptors” for that effector moiety. For example,where IFNα is the effector moiety, the effector moiety receptor thatbinds to an IFNα molecule (e.g. an antibody fused to IFNα) is the IFNαreceptor 1 or 2 (see UniProt accession no. P17181 (version 121) and NCBIRefSeq NP_(—)000620.2 for human IFNα receptor 1, and UniProt accessionno. P48551 (version 131) and NCBI RefSeqs NP_(—)997467.1 &NP_(—)997468.1 for human IFNα receptor 2).

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. AnIgG class antibody essentially consists of two Fab fragments and an Fcdomain, linked via the immunoglobulin hinge region.

As used herein, “Fab fragment” refers to an antibody fragment comprisinga light chain fragment comprising a VL domain and a constant domain of alight chain (CL), and a VH domain and a first constant domain (CH1) of aheavy chain.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g. Kindt etal., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91 (2007).A single VH or VL domain may be sufficient to confer antigen-bindingspecificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196, 901-917 (1987)). Exemplary CDRs (CDR-L1,CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and95-102 of H3 (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). With the exception of CDR1 in VH, CDRs generallycomprise the amino acid residues that form the hypervariable loops. CDRsalso comprise “specificity determining residues,” or “SDRs,” which areresidues that contact antigen. SDRs are contained within regions of theCDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1,a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at aminoacid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58of H2, and 95-102 of H3 (see Almagro and Fransson, Front. Biosci. 13,1619-1633 (2008)). Unless otherwise indicated, HVR residues and otherresidues in the variable domain (e.g. FR residues) are numbered hereinaccording to Kabat et al., supra (refered to as “Kabat numbering”).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “parent antibody” herein refers to an antibody that serves as astarting point or basis for the preparation of an antibody variant. Inone embodiment, the parent antibody is a humanized or human antibody.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. An IgG Fc region comprises an IgG CH2and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usuallyextends from an amino acid residue at about position 231 to an aminoacid residue at about position 340. In one embodiment, a carbohydratechain is attached to the CH2 domain. The CH2 domain herein may be anative sequence CH2 domain or variant CH2 domain. The “CH3 domain”comprises the stretch of residues C-terminal to a CH2 domain in an Fcregion (i.e. from an amino acid residue at about position 341 to anamino acid residue at about position 447 of an IgG). The CH3 regionherein may be a native sequence CH3 domain or a variant CH3 domain (e.g.a CH3 domain with an introduced “protroberance” (“knob”) in one chainthereof and a corresponding introduced “cavity” (“hole”) in the otherchain thereof; see U.S. Pat. No. 5,821,333, expressly incorporatedherein by reference). Such variant CH3 domains may be used to promoteheterodimerization of two non-identical antibody heavy chains as hereindescribed. In one embodiment, a human IgG heavy chain Fc region extendsfrom Cys226, or from Pro230, to the carboxyl-terminus of the heavychain. However, the C-terminal lysine (Lys447) of the Fc region may ormay not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637 (version 141)).

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2-20 amino acids. Peptide linkers are knownin the art or are described herein. Suitable, non-immunogenic linkerpeptides include, for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n)peptide linkers. “n” is generally a number between 1 and 10, typicallybetween 2 and 4.

A “modification promoting heterodimerization” is a manipulation of thepeptide backbone or the post-translational modifications of apolypeptide, e.g. an antibody heavy chain, that reduces or prevents theassociation of the polypeptide with an identical polypeptide to form ahomodimer. A modification promoting heterodimerization as used hereinparticularly includes separate modifications made to each of twopolypeptides desired to form a dimer, wherein the modifications arecomplementary to each other so as to promote association of the twopolypeptides. For example, a modification promoting heterodimerizationmay alter the structure or charge of one or both of the polypeptidesdesired to form a dimer so as to make their association sterically orelectrostatically favorable, respectively. Heterodimerization occursbetween two non-identical polypeptides, such as two antibody heavychains wherein further components attached to each of the heavy chains(e.g. effector moiety) are not the same. In the antibodies according tothe present invention, the modification promoting heterodimerization isin the Fc domain, particularly in the CH3 domain. In some embodimentsthe modification promoting heterodimerziation comprises an amino acidmutation, specifically an amino acid substitution. In a particularembodiment, the modification promoting heterodimerization comprises aseparate amino acid mutation, specifically an amino acid substitution,in each of the two antibody heavy chains.

A “knob-into-hole modification” refers to a modification within theinterface between two antibody heavy chains in the CH3 domain, whereini) in the CH3 domain of one heavy chain, an amino acid residue isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance (“knob”) within the interface in theCH3 domain of one heavy chain which is positionable in a cavity (“hole”)within the interface in the CH3 domain of the other heavy chain, and ii)in the CH3 domain of the other heavy chain, an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity (“hole”) within the interface in the secondCH3 domain within which a protuberance (“knob”) within the interface inthe first CH3 domain is positionable. In one embodiment, the“knob-into-hole modification” comprises the amino acid substitutionT366W and optionally the amino acid substitution S354C in one of theantibody heavy chains, and the amino acid substitutions T366S, L368A,Y407V and optionally Y349C in the other one of the antibody heavychains.

An amino acid “substitution” refers to the replacement in a polypeptideof one amino acid with another amino acid. In one embodiment, an aminoacid is replaced with another amino acid having similar structuraland/or chemical properties, e.g., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Non-conservative substitutions will entail exchanging amember of one of these classes for another class. For example, aminoacid substitutions can also result in replacing one amino acid withanother amino acid having different structural and/or chemicalproperties, for example, replacing an amino acid from one group (e.g.,polar) with another amino acid from a different group (e.g., basic).Amino acid substitutions can be generated using genetic or chemicalmethods well known in the art. Genetic methods may include site-directedmutagenesis, PCR, gene synthesis and the like. It is contemplated thatmethods of altering the side chain group of an amino acid by methodsother than genetic engineering, such as chemical modification, may alsobe useful. Various designations may be used herein to indicate the sameamino acid substitution. For example, a substitution from proline atposition 329 of the Fc domain to glycine can be indicated as 329G, G329,G₃₂₉, P329G, or Pro329Gly.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

“Polynucleotide” or “nucleic acid” as used interchangeably herein,refers to polymers of nucleotides of any length, and include DNA andRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase or by asynthetic reaction. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and their analogs. A sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may comprise modification(s) made after synthesis, suchas conjugation to a label.

The term “vector” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe antibodies of the present invention. Host cells include culturedcells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PERcells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, andplant cells, to name only a few, but also cells comprised within atransgenic animal, transgenic plant or cultured plant or animal tissue.An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

Antibodies of the Invention

The invention provides novel antibodies, particularly monoclonalantibodies, that bind to the asialoglycoprotein receptor (ASGPR).

In a first aspect, the invention provides an antibody capable ofspecific binding to asialoglycoprotein receptor (ASGPR), wherein theantibody comprises a) the heavy chain variable region sequence of SEQ IDNO: 16 and the light chain variable region sequence of SEQ ID NO: 14; b)the heavy chain variable region sequence of SEQ ID NO: 4 and the lightchain variable region sequence of SEQ ID NO: 2; c) the heavy chainvariable region sequence of SEQ ID NO: 8 and the light chain variableregion sequence of SEQ ID NO: 6; d) the heavy chain variable regionsequence of SEQ ID NO: 12 and the light chain variable region sequenceof SEQ ID NO: 10; e) the heavy chain variable region sequence of SEQ IDNO: 20 and the light chain variable region sequence of SEQ ID NO: 18; f)the heavy chain variable region sequence of SEQ ID NO: 24 and the lightchain variable region sequence of SEQ ID NO: 22; g) the heavy chainvariable region sequence of SEQ ID NO: 28 and the light chain variableregion sequence of SEQ ID NO: 26; h) the heavy chain variable regionsequence of SEQ ID NO: 4 and the light chain variable region sequence ofSEQ ID NO: 30; i) the heavy chain variable region sequence of SEQ ID NO:4 and the light chain variable region sequence of SEQ ID NO: 32; j) theheavy chain variable region sequence of SEQ ID NO: 4 and the light chainvariable region sequence of SEQ ID NO: 34; or k) the heavy chainvariable region sequence of SEQ ID NO: 24 and the light chain variableregion sequence of SEQ ID NO: 22.

A particular antibody according to the invention comprises the heavychain variable region sequence of SEQ ID NO: 16 and the light chainvariable region sequence of SEQ ID NO: 14. This antibody clone isdesignated 4F3. Another particular antibody according to the inventioncomprises the heavy chain variable region sequence of SEQ ID NO: 4 andthe light chain variable region sequence of SEQ ID NO: 2. This antibodyis designated 51A12.

The invention provides antibodies which are capable of specific bindingto ASGPR and compete for binding to an epitope of ASGPR with antibody4F3. In one embodiment, such an antibody binds to the same epitope as4F3. The 4F3 antibody recognizes an epitope in the stalk region ofASGPR. Accordingly, in one embodiment, such an antibody recognizes anepitope in the stalk region of ASGPR. Also contemplated by the inventionare affinity matured variants of the 4F3 antibody. In one embodiment,such an antibody comprises a heavy chain variable region sequence thatis at least about 96%, 97%, 98% or 99% identical to the sequence of SEQID NO: 16, and a light chain variable region sequence that is at leastabout 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 14.In one embodiment, such an antibody comprises the light chain variableregion sequence of SEQ ID NO: 14 with one, two, three, four, five, sixor seven, particularly two, three, four or five, amino acidsubstitutions. In one embodiment, such an antibody comprises the heavychain variable region sequence of SEQ ID NO: 16 with one, two, three,four, five, six or seven, particularly two, three, four or five, aminoacid substitutions. Variants of the 4F3 antibody may also comprise aheavy chain variable region which is identical to the heavy chainvariable region of 4F3, together with a variant light chain variableregion, or vice versa.

The invention further provides antibodies which are capable of specificbinding to ASGPR and compete for binding to an epitope of ASGPR withantibody 51A12. In one embodiment, such and antibody binds to the sameepitope as 51A12. The 51A12 antibody recognizes and epitope in thecarbohydrate recognition domain (CRD) of ASGPR. Accordingly, in oneembodiment, such an antibody recognizes an epitope in the CRD of ASGPR.Also contemplated by the invention are affinity matured variants of the51A12 antibody, particularly variants obtained by randomization of thelight chain CDR3 or 51A12. In one embodiment, such an antibody comprisesa heavy chain variable region sequence that is at least about 96%, 97%,98% or 99% identical to the sequence of SEQ ID NO: 4, and a light chainvariable region sequence that is at least about 96%, 97%, 98% or 99%identical to the sequence of SEQ ID NO: 2. In one embodiment, such anantibody comprises the light chain variable region sequence of SEQ IDNO: 2 with one, two, three, four, five, six or seven, particularly two,three, four or five, amino acid substitutions. In one embodiment, suchan antibody comprises the heavy chain variable region sequence of SEQ IDNO: 4 with one, two, three, four, five, six or seven, particularly two,three, four or five, amino acid substitutions. Variants of the 51A12antibody may also comprise a heavy chain variable region which isidentical to the heavy chain variable region of 51A12, together with avariant light chain variable region, or vice versa. In one embodiment,such an antibody comprises a) the heavy chain variable region sequenceof SEQ ID NO: 4 and the light chain variable region sequence of SEQ IDNO: 36; b) the heavy chain variable region sequence of SEQ ID NO: 4 andthe light chain variable region sequence of SEQ ID NO: 38; c) the heavychain variable region sequence of SEQ ID NO: 8 and the light chainvariable region sequence of SEQ ID NO: 40; d) the heavy chain variableregion sequence of SEQ ID NO: 12 and the light chain variable regionsequence of SEQ ID NO: 42; e) the heavy chain variable region sequenceof SEQ ID NO: 20 and the light chain variable region sequence of SEQ IDNO: 44; f) the heavy chain variable region sequence of SEQ ID NO: 24 andthe light chain variable region sequence of SEQ ID NO: 46; or g) theheavy chain variable region sequence of SEQ ID NO: 28 and the lightchain variable region sequence of SEQ ID NO: 48.

Preferably, the antibodies of the invention are human antibodies, i.e.the antibodies comprise human variable and constant regions. In oneembodiment, the antibodies comprise a human Fc region, particularly ahuman IgG Fc region, more particularly a human IgG₁ Fc region.Particular antibodies according to the invention are full-lengthantibodies, particularly full-length IgG class antibodies, moreparticularly full-length IgG₁ subclass antibodies. Alternatively, theantibodies may be antibody fragments. In one embodiment, the antibodiesare Fab fragments or scFv fragments. In some embodiments, the antibodiescomprise a Fab fragment and an Fc region, particularly a human IgG Fcregion, more particularly a human IgG₁ Fc region, linked by animmunoglobulin hinge region, particularly a human IgG hinge region, moreparticularly a human IgG₁ hinge region. Specifically, the antibodies maycomprise a Fab fragment and an Fc region, linked by an immunoglobulinhinge region, wherein no further Fab fragment is present. In suchembodiments, the antibodies are essentially full-length antibodies,lacking one Fab fragment.

Fc regions comprised in the antibodies of the invention may comprisevarious modifications, as compared to a native Fc region.

While the Fc domain confers to the antibodies favorable pharmacokineticproperties, including a long serum half-life which contributes to goodaccumulation in the target tissue and a favorable tissue-blooddistribution ratio, it may at the same time lead to undesirabletargeting of the antibodies to cells expressing Fc receptors rather thanto the preferred antigen-bearing cells. Moreover, the co-activation ofFc receptor signaling pathways may lead to cytokine release which,particularly in antibodies having an effector moiety (e.g. a cytokine)attached, results in excessive activation of cytokine receptors andsevere side effects upon systemic administration. Therefore, in someembodiments, the antibodies comprise in the Fc region a modificationreducing binding affinity of the antibody to an Fc receptor,particularly an Fcγ receptor, as compared to a corresponding antibodycomprising an unmodified Fc region. Binding to Fc receptors can beeasily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR)using standard instrumentation such as a BIAcore instrument (GEHealthcare) and Fc receptors such as may be obtained by recombinantexpression. A specific illustrative and exemplary embodiment formeasuring binding affinity is described in the following. According toone embodiment, Binding affinity to an Fc receptor is measured bysurface plasmon resonance using a BIACORE® T100 machine (GE Healthcare)at 25° C. with ligand (Fc receptor) immobilized on CM5 chips. Briefly,carboxymethylated dextran biosensor chips (CM5, GE Healthcare) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Recombinant ligand is diluted with 10 mM sodiumacetate, pH 5.5, to 0.5-30 μg/ml before injection at a flow rate of 10μl/min to achieve approximately 100-5000 response units (RU) of coupledprotein. Following the injection of the ligand, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, three- tofive-fold serial dilutions of antibody (range between ˜0.01 nM to 300nM) are injected in HBS-EP+ (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate ofapproximately 30-50 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® T100 Evaluation Software version 1.1.1) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).Alternatively, binding affinity antibodies to Fc receptors may beevaluated using cell lines known to express particular Fc receptors,such as NK cells expressing FcγIIIa receptor.

In the modification comprises one or more amino acid mutation thatreduces the binding affinity of the antibody to an Fc receptor.Typically, the same one or more amino acid mutation is present in eachof the two antibody heavy chains in the Fc domain. In one embodimentsaid amino acid mutation reduces the binding affinity of the antibody tothe Fc receptor by at least 2-fold, at least 5-fold, or at least10-fold. In embodiments where there is more than one amino acid mutationthat reduces the binding affinity of the antibody to the Fc receptor,the combination of these amino acid mutations may reduce the bindingaffinity of the antibody to the Fc receptor by at least 10-fold, atleast 20-fold, or even at least 50-fold. In one embodiment the antibodyexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared to anantibody comprising an unmodified Fc domain.

In one embodiment the Fc receptor is an activating Fc receptor. In aspecific embodiment the Fc receptor is an Fcγ receptor, morespecifically an FcγRIIIa, FcγRI or FcγRIIa receptor. Preferably, bindingto each of these receptors is reduced. In some embodiments bindingaffinity to a complement component, specifically binding affinity toC1q, is also reduced. In one embodiment binding affinity to neonatal Fcreceptor (FcRn) is not reduced. Substantially similar binding to FcRn,i.e. preservation of the binding affinity of the antibody to saidreceptor, is achieved when the antibody exhibits greater than about 70%of the binding affinity of an unmodified form of the antibody to FcRn.Antibodies of the invention may exhibit greater than about 80% and evengreater than about 90% of such affinity. In one embodiment the aminoacid mutation is an amino acid substitution. In one embodiment theantibody comprises an amino acid substitution in the Fc region atposition P329 (EU numbering). In a more specific embodiment the aminoacid substitution is P329A or P329G, particularly P329G. In oneembodiment the antibody comprises a further amino acid substitution inthe Fc region at a position selected from 5228, E233, L234, L235, N297and P331. In a more specific embodiment the further amino acidsubstitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment the antibody comprises amino acidsubstitutions in the Fc region at positions P329, L234 and L235. In amore particular embodiment the antibody comprises the amino acidmutations L234A, L235A and P329G (LALA P329G). This combination of aminoacid substitutions almost completely abolishes Fcγ receptor binding of ahuman IgG antibody, as described in PCT patent application no.PCT/EP2012/055393, incorporated herein by reference in its entirety. PCTpatent application no. PCT/EP2012/055393 also describes methods ofpreparing such modified antibodies and methods for determining itsproperties such as Fc receptor binding or effector functions.

Antibodies comprising modifications in the Fc region can be prepared byamino acid deletion, substitution, insertion or modification usinggenetic or chemical methods well known in the art. Genetic methods mayinclude site-specific mutagenesis of the encoding DNA sequence, PCR,gene synthesis, and the like. The correct nucleotide changes can beverified for example by sequencing.

Antibodies which comprise modifications reducing Fc receptor bindinggenerally have reduced effector functions, particularly reduced ADCC, ascompared to corresponding unmodified antibodies. In some embodiments theantibodies have reduced ADCC. In specific embodiments the reduced ADCCis less than 20% of the ADCC induced by a corresponding antibodycomprising an unmodified Fc region. Effector function of an antibody canbe measured by methods known in the art. Examples of in vitro assays toassess ADCC activity of a molecule of interest are described in U.S.Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med166, 1351-1361 (1987). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.); andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison,Wis.)). Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g. in a animal model such as that disclosed inClynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). In someembodiments binding of the antibody to a complement component,specifically to C1q, is also reduced. Accordingly, complement-dependentcytotoxicity (CDC) may also be reduced. C1q binding assays may becarried out to determine whether the antibody is able to bind C1q andhence has CDC activity. See e.g. C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052(2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

In addition to the antibodies described hereinabove and in PCT patentapplication no. PCT/EP2012/055393, antibodies with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments, the antibodies of the invention are IgG₄ subclassantibodies, particularly human IgG₄ subclass antibodies. In oneembodiment the IgG₄ antibody comprises amino acid substitutions in theFc region at position S228, specifically the amino acid substitutionS228P. To further reduce its binding affinity to an Fc receptor and/orits effector function, in one embodiment the IgG₄ antibody comprises anamino acid substitution at position L235, specifically the amino acidsubstitution L235E. In another embodiment, the IgG₄ antibody comprisesan amino acid substitution at position P329, specifically the amino acidsubstitution P329G. In a particular embodiment, the IgG₄ antibodycomprises amino acid substitutions at positions S228, L235 and P329,specifically amino acid substitutions S228P, L235E and P329G. Suchmodified IgG₄ antibodies and their Fey receptor binding properties aredescribed in PCT patent application no. PCT/EP2012/055393, incorporatedherein by reference in its entirety.

Antibodies according to the invention may have effector moieties such ascytokines attached. In particular embodiments, the antibodies compriseonly one single effector moiety, fused to one of the two antibody heavychains, thus these antibodies comprise two non-identical polypeptidechains. Similarly, the antibodies of the invention may be full-lengthantibodies, lacking one of the Fab fragments, hence comprising a fullantibody heavy chain and an antibody heavy chain lacking the VH and CH1domains. Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides, out of which only heterodimers of the two non-identicalpolypeptides are useful. To improve the yield and purity of suchantibodies in recombinant production, it can thus be advantageous tointroduce in the Fc region of the antibody a modification which hindersthe formation of homodimers of two identical polypeptides (e.g. twopolypeptides comprising an effector moiety, or two polypeptides lackingan effector moiety) and/or promotes the formation of heterodimers of apolypeptide comprising an effector moiety and a polypeptide lacking aneffector moiety. Accordingly, in some embodiments, the antibodies of theinvention comprise in the Fc region a modification promotingheterodimerization of two non-identical antibody heavy chains. The siteof most extensive protein-protein interaction between the two heavychains of a human IgG antibody is in the CH3 domain of the Fc region.Thus, in one embodiment said modification is in the CH3 domain of the Fcregion. In a specific embodiment, said modification is a knob-into-holemodification, comprising a knob modification in one of the antibodyheavy chains and a hole modification in the other one of the twoantibody heavy chains. The knob-into-hole technology is described e.g.in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al.,Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).Generally, the method involves introducing a protuberance (“knob”) atthe interface of a first polypeptide and a corresponding cavity (“hole”)in the interface of a second polypeptide, such that the protuberance canbe positioned in the cavity so as to promote heterodimer formation andhinder homodimer formation. Protuberances are constructed by replacingsmall amino acid side chains from the interface of the first polypeptidewith larger side chains (e.g. tyrosine or tryptophan). Compensatorycavities of identical or similar size to the protuberances are createdin the interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). Hence, in oneembodiment, the antibody comprises a modification within the interfacebetween the two antibody heavy chains in the CH3 domain, wherein i) inthe CH3 domain of one heavy chain, an amino acid residue is replacedwith an amino acid residue having a larger side chain volume, therebygenerating a protuberance (“knob”) within the interface in the CH3domain of one heavy chain which is positionable in a cavity (“hole”)within the interface in the CH3 domain of the other heavy chain, and ii)in the CH3 domain of the other heavy chain, an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity (“hole”) within the interface in the secondCH3 domain within which a protuberance (“knob”) within the interface inthe first CH3 domain is positionable. The protuberance and cavity can bemade by altering the nucleic acid encoding the polypeptides, e.g. bysite-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W (EU numbering) in one of the two antibody heavy chains, and thehole modification comprises the amino acid substitutions T366S, L368Aand Y407V (EU numbering) in the other one of the two antibody heavychains. In a further specific embodiment, the antibody heavy chaincomprising the knob modification additionally comprises the amino acidsubstitution S354C, and the antibody heavy chain comprising the holemodification additionally comprises the amino acid substitution Y349C.Introduction of these two cysteine residues results in formation of adisulfide bridge between the two antibody heavy chains in the Fc region,further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15(2001)). In an alternative embodiment a modification promotingheterodimerization of two non-identical antibody heavy chains comprisesa modification mediating electrostatic steering effects, e.g. asdescribed in PCT publication WO 2009/089004. Generally, this methodinvolves replacement of one or more amino acid residues at the interfaceof the two antibody heavy chains by charged amino acid residues so thathomodimer formation becomes electrostatically unfavorable butheterodimerization electrostatically favorable. In a particularembodiment wherein the antibody has an effector moiety attached to it,the effector moiety is fused to the amino- or carboxy-terminal aminoacid of the antibody heavy chain comprising the knob modification.Without wishing to be bound by theory, fusion of the effector moiety tothe knob-containing heavy chain will further minimize the generation ofhomodimeric antibodies comprising two effector moieties (steric clash oftwo knob-containing polypeptides). Similarly, in embodiments wherein theantibody comprises a only single Fab fragment fused to an Fc region, theFab fragment is preferably fused to the heavy chain of the Fc regioncomprising the knob modification.

The antibodies of the invention combine a number of properties which areparticularly advantageous, for example for therapeutic applications. Forexample, the antibodies are cross-reactive for human and cynomolgusmonkey, which enables e.g. in vivo studies in cynomolgus monkeys priorto human use. Hence, in one embodiment, the antibody of the invention iscapable of specific binding to human and cynomolgus monkey ASGPR.Furthermore, the antibodies of the invention bind ASGPR withparticularly strong affinity and/or avidity. In one embodiment, theantibody binds to human ASGPR with an dissociation constant (K_(D)) ofsmaller than 1 μM, particularly smaller than 100 nM, more particularlysmaller than 1 nM, when measured as Fab fragment by Surface PlasmonResonance (SPR). A method for measuring binding affinity by SPR isdescribed herein. Specifically, measurement is made at a temperature of25° C. In one embodiment, affinity (K_(D)) of antibodies as Fabfragements is measured by SPR using a ProteOn XPR36 instrument (Biorad)at 25° C. with biotinylated mono- (avi-Fc-human ASGPR H1 CRD, SEQ ID NO:118) or bivalent (avi-Fc-human ASGPR H1 stalk-CRD, SEQ ID NO: 130) ASGPRH1 antigens immobilized on NLC chips by neutravidin capture. In anexemplary method, antigens for immobilization are diluted with PBST (10mM phosphate, 150 mM NaCl pH 7.4, 0.005% Tween-20) to 10 μg/ml, andinjected at 30 μl/min at varying contact times, to achieveimmobilization levels of 200, 400 or 800 response units (RU) in verticalorientation. Subsequently, analytes (antibodies) are injected. Forone-shot kinetics measurements, injection direction is changed tohorizontal orientation, and two-fold dilution series of purified Fabfragments (varying concentration ranges between 100 and 6.25 nM) areinjected simultaneously at 50, 60 or 100 μl/min along separate channels1-5, with association times of 150 or 200 s, and dissociation times of240 or 600 s. Buffer (PBST) is injected along the sixth channel toprovide an “in-line” blank for referencing. Association rate constants(k_(on)) and dissociation rate constants (k_(off)) are calculated usinga simple one-to-one Langmuir binding model in ProteOn Manager v3.1software by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) is calculatedas the ratio k_(off)/k_(on). Regeneration is performed in horizontalorientation using 10 mM glycine, pH 1.5 at a flow rate of 100 μl/min fora contact time of 30 s. In another embodiment, the antibody binds tohuman ASGPR with a K_(D) of smaller than 1 μM, particularly smaller than500 nM, more particularly smaller than 100 nM or even smaller than 10nM, when measured as IgG₁ by fluoresecence resonance energy transfer(FRET). A method for measuring binding affinity (or avidity) by FRET isdescribed herein. In one embodiment, the measurement is performed bycontacting cells expressing full-length ASGPR protein labeled with aFRET donor molecule with the antibodies, and detection of boundantibodies by a secondary antibody labeled with a suitable FRET acceptormolecule. In an exemplary method, the DNA sequence encoding for the SNAPTag (plasmid purchased from Cisbio) is amplified by PCR and ligated intoan expression vector, containing the full length human ASGPR H1 sequence(Origene). The resulting fusion protein is comprised of full-lengthASGPR H1 with a C-terminal SNAP tag. HEK293 cells are transfected with10 μg DNA using Lipofectamine 2000 as transfection reagent. After anincubation time of 20 h, cells are washed with PBS and incubated for 1 hat 37° C. in LabMed buffer (Cisbio) containing 100 nM SNAP-Lumi4Tb(Cibsio), leading to specific labeling of the SNAP Tag. Subsequently,cells are washed 4 times with LabMed buffer to remove unbound dye. Thelabeling efficiency is determined by measuring the emission of terbiumat 615 nm compared to buffer. Cells can then be stored frozen at −80° C.for up to 6 months. Avidity is measured by adding ASGPR-specificantibodies at a concentration ranging from 50-0.39 nM to labeled cells(100 cells per well) followed by addition of anti-human Fc-d2 (Cisbio,final 200 nM per well) as acceptor molecule for the FRET. After anincubation time of 3 h at RT the emission of the acceptor dye (665 nm)as well as of the donor dye (615 nm) is determined using a fluorescenceReader (Victor 3, Perkin Elmer). The ratio of acceptor to donor emissionis calculated and the ratio of the background control (cells withanti-human Fc-d2) subtracted. Curves can be analysed in GraphPad Prism5software and K_(D) values calculated. A further advantage of theantibodies of the invention is that they do not compete for binding toASGPR with natural ligands of the receptor (asialoglycoproteins such ase.g. asialofetuin), i.e. antibody binding is not affected by thepresence of ASGPR ligand and does not interfere with the naturalfunction of ASGPR. In one embodiment, the antibody does not compete witha natural ligand of ASGPR for binding to ASGPR. In a specificembodiment, said natural ligand of ASGPR is asialofetuin. Competitioncan be measured by methods well known in the art. In one embodiment,competition with a natural ligand of ASGPR is measured by FACS, forexample using an ASGPR-expressing cell line, fluorescently labeledligand, and detection of bound antibodies with a secondary antibodyhaving a different fluorescent label. In an exemplary method, thehepatocellular carcinoma cell line HepG2 is used. 0.2 mio cells per wellin a 96 well round bottom plate are incubated with 40 μl of Alexa488labeled asialofetuin (from fetal calf serum, Sigma Aldrich #A4781, finalconcentration 100 μg/ml) at 4° C. for 30 min. The binding is performedin the presence of calcium, as ligand binding to ASGPR is calciumdependent. Unbound protein is removed by washing the cells once withHBSS containing 0.1% BSA. Then 40 μl of the anti-ASGPR antibodies (30,6, and 1.25 μg/ml final concentration) are added to the cells in thepresence of 100 μg/ml asialofetuin. Cells are incubated for 30 min at 4°C. and unbound protein is removed by washing the cells once. AnAPC-conjugated AffiniPure goat anti-human IgG Fc gamma fragment-specificsecondary F(ab′)2 fragment (Jackson Immuno Research #109-136-170;working solution 1:50 in HBSS containing 0.1% BSA) is used as ansecondary antibody. After 30 min incubation at 4° C. unbound secondaryantibody is removed by washing. Cells are fixed using 1% PFA and bindingof ligand as well as antibodies is analyzed using BD FACS Cantoll(Software BD DIVA). A major advantage of the antibodies of the inventionis their high specificity for ASGPR. For example, despite their strongbinding to human ASGPR H1, the antibodies do not detectably bind toCLEC10A, which was identified as the closest homologue of human ASGPRH1. In one embodiment, the antibody does not detectably bind to CLEC10A,particularly human CLEC10A. Specifically, the antibody does notdetectably bind to CLEC10A wherein binding is measured by SPR (asdescribed herein). Moreover, the antibodies bind to cells which do notexpress ASGPR only to a similar extent as a corresponding untargetedantibody (isotype control). Hence, in one embodiment, the antibody doesnot specifically bind to cells lacking ASGPR expression, particularlyhuman cells, more particularly human blood cells. Exemplary cellslacking ASGPR expression include Hela cells (a human cell line derivedfrom cervical cancer), A459 human non-small cell lung cancer cells,human embryonic kidney (HEK) cells, and (human) PBMCs. Binding, or lackof binding, to specific cells can easily be determined for example byFACS. Such methods are well established in the art and also described inthe Examples hereinbelow. An important feature of anti-ASGPR antibodiesare their internalization characteristics. For example, if the antibodyis to be used to target an effector moiety to ASGPR-expressing cells, itis desirable for the antibody to be present on the cell surface for asufficiently long time for activation of effector moiety receptors. Theantibodies of the present invention, upon binding to ASGPR, areinternalized into the ASGPR-expressing cell, however, they are recycledback to the cell surface without being degraded inside the cell. Hence,in one embodiment, the antibody is internalized into a cell expressingASGPR upon binding of the antibody to ASGPR on the surface of said cell.In a specific embodiment, the antibody is recycled back to the surfaceof said cell at about the same rate as it is internalized into saidcell. Internalization and recycling of cell surface proteins orantibodies bound thereto can easily be measured by established methods,such as FACS or (confocal) microscopy techniques. In one embodiment,internalization and/or recycling are measured by FACS. For an antibodyto have sustained effects, it is important that its target antigen ispresent at essentially constant levels. Frequently, antibody binding totarget antigens induces downregulation of the latter, leading to reducedefficacy of antibodies. However, the antibodies of the present inventiondo not have such effect. In one embodiment, the antibody does notsignificantly induce downregulation of ASGPR expression at the surfaceof a cell upon binding of the antibody to ASGPR on the surface of saidcell. The level of antigen expression at the surface of a cell caneasily be determined by established methods such as FACS.

Antibodies with Attached Effector Moieties

Particularly useful antibodies according to the present invention areantibodies having an effector moiety, e.g. a cytokine, attached.Antibodies fused to an effector moiety such as a cytokine are alsoreferred to as immunoconjugates herein. The antibodies with attachedeffector moieties can incorporate, singly or in combination, any of thefeatures described hereinabove in relation to the antibodies of theinvention.

Accordingly, in one aspect, the invention provides an antibody capableof specific binding to ASGPR according to any of the above embodiments,wherein an effector moiety is attached to the antibody. In oneembodiment, not more than one effector moiety is attached to theantibody. The absence of further effector moieties may reduce targetingof the antibody to sites where the respective effector moiety receptoris presented, thereby improving targeting to and accumulation at siteswhere the actual target antigen of the antibody, ASGPR, is presented.Furthermore, the absence of an avidity effect for the respectiveeffector moiety receptor can reduce activation of effector moietyreceptor-positive cells in peripheral blood upon intravenousadministration of the antibody. The effector moieties for use in theinvention are generally polypeptides that influence cellular activity,for example, through signal transduction pathways. Accordingly, aneffector moiety useful in the invention can be associated withreceptor-mediated signaling that transmits a signal from outside thecell membrane to modulate a response within the cell. For example, aneffector moiety can be a cytokine. In particular embodiments theeffector moiety is human. In particular embodiments, the effector moietyis a peptide molecule and is fused to the antibody through peptide bonds(i.e. the antibody and effector moiety form a fusion protein). In oneembodiment, the effector moiety is single-chain peptide molecule. In afurther embodiment, the effector moiety is fused at its amino-terminalamino acid to the carboxy-terminal amino acid of one of the heavy chainsof the antibody, optionally through a peptide linker. Suitable,non-immunogenic peptide linkers include, for example, (G₄S)_(n),(SG₄)_(n) or G₄(SG₄)_(n) peptide linkers. “n” is generally a numberbetween 1 and 10, typically between 2 and 4. In embodiments wherein theantibody comprises a knob-into-hole modification in the Fc region asdescribed above, it is preferable to fuse the effector moiety to theantibody heavy chain comprising the knob modification.

In one embodiment said effector moiety is a cytokine molecule. Examplesof useful cytokines include, but are not limited to, GM-CSF, IL-1α,IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-21,IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. In oneembodiment, said cytokine molecule is fused at its amino-terminal aminoacid to the carboxy-terminal amino acid of one of the antibody heavychains, optionally through a peptide linker. In one embodiment, saidcytokine molecule is a human cytokine. In one embodiment, said cytokinemolecule is an interferon molecule. In a specific embodiment, saidinterferon molecule is interferon alpha, particularly human interferonalpha, more particularly human interferon alpha 2 (see SEQ ID NO: 138)or human interferon alpha 2a (see SEQ ID NO: 139). Interferon alpha isknown to have anti-viral activities. Hence, attaching an interferonmolecule to an antibodiy of the invention is particularly useful fortargeting virus-infected ASGPR-expressing cells. In one embodiment,wherein the cytokine molecule is an interferon molecule, the antibodyhas anti-viral activity in cells expressing ASGPR on their surface. Inone embodiment, said cells are liver cells, particularly hepatocytes,more particularly human hepatocytes. In one embodiment, said anti-viralactivitiy is selective. In a specific embodiment, the antibody has noanti-viral activity in cells not expressing significant levels of ASGPRon their surface. In one embodiment, said cells are blood cells,particularly human blood cells. In one embodiment, said anti-viralactivity This selectivity of interferon molecules attached to anti-ASGPRantibodies according to the invention is in contrast to untargetedinterferon molecules, which do not distinguish between any intendedtarget cells (e.g. hepatocytes) and cells which should not be affected(e.g. blood cells), and is crucial for possible therapeutic use withoutmajor toxicity issues. In a further specific embodiment, said anti-viralactivity is selected from inhibition of viral infection, inhibition ofvirus replication, inhibition of cell killing and induction of one ormore interferon-stimulated gene. In a specific embodiment, the one ormore interferon-stimulated gene is selected from the group of MX1(myxovirus restistance 1, also known as interferon-induced protein p78),RSAD2 (radical S-adenosyl methionine domain containing 2, also known ascytomegalovirus-induced gene 5), HRASLS2 (HRAS-like suppressor 2), IFIT1(interferon-induced protein with tetratricopeptide repeats 1), andIFITM2 (interferone-induced transmembrane protein 2). In one embodiment,the induction of one or more interferon-stimulated gene is at least a1.5-fold, particularly at least a 2-fold, more particularly at least a5-fold induction on mRNA level, as compared to induction by acorresponding antibody without interferon molecule attached. Geneinduction on mRNA level can be measured by methods well established inthe art, including quantitative reverse-transcription (RT) PCR ormicroarray analysis, as described herein. Inhibition of cell killing canbe determined for example by a virus protection assay, wherein cells arepreincubated with the test compound, followed by addition of virus andquantification of living cells after incubation. An exemplary such assayis described in the Examples. Madin-Darby bovine kidney (MDBK) cells arepre-incubated with the antibodies and controls for 1-4 h. Vesicularstomatitis virus is then added for additional 16-24 h. At the end ofthis incubation step, living cells are stained with crystal violetstaining solution (0.5%) and quantification of living cells is performedusing a microplate reader at 550-600 nm with a reference wavelength of690 nm. An exemplary assay for assessment of virus replication is alsoprovided in the Examples. This assay uses a Huh 7-derivedhepatocarcinoma cell line stably transfected with bicistronic hepatitisC virus (HCV) replicon of which the first open reading frame, driven bythe HCV IRES, contains the renilla luciferase gene in fusion with theneomycin phosphotransferase gene (NPTII) and the second open readingframe, driven by EMCV IRES, contains the HCV non-structural genes NS3,NS4a, NS4b, NS5A and NS5B derived from the NK5.1 replicon backbone.Cells are cultured at 37° C. in a humidified atmosphere with 5% CO₂ inDMEM supplemented with Glutamax™ and 100 mg/ml sodium pyruvate. Themedium was further supplemented with 10% (v/v) FBS (v/v)penicillin/streptomycin and 1% (v/v) geneticin. The cells in DMEMcontaining 5% (v/v) FBS are plated in 96-well plates at 5000 cells/wellin 90 μl volume. 24 hours after plating, antibodies (or medium as acontrol) are added to the cells in 3-fold dilutions over 12 wells(0.01-2000 pM), in a volume of 10 μl, so that the final volume afteraddition of antibody is 100 μl. Renilla luciferase reporter signal isread 72 hours after adding antibodies, using the Renilla LuciferaseAssay system (Promega, # E2820). The EC₅₀ values are calculated as thecompound concentration at which a 50% reduction in the level of renillaluciferase reporter is observed as compared to control samples (in theabsence of antibody). Dose-response curves and EC₅₀ values are obtainedby using the XLfit4 program (ID Business Solutions Ltd., Surrey, UK). Ina particular embodiment, the antibody according to the invention is afull-length human IgG₁ antibody comprising the heavy chain variableregion sequence of SEQ ID NO: 16 and the light chain variable regionsequence of SEQ ID NO: 14, said antibody comprising in the Fc region amodification reducing binding affinity of the antibody to FcγRIIIa and aknob-into-hole modification, said knob-into-hole modification comprisinga knob modification in one of the antibody heavy chains and a holemodification in the other one of the antibody heavy chains, and saidantibody having an IFNα2 molecule fused at the N-terminal amino acid tothe C-terminal amino acid of one of the antibody heavy chains through apeptide linker. In a specific embodiment, said modification reducingbinding affinity of the antibody to FcγRIIIa comprises the amino acidsubstitutions L234A, L235A and P329G (EU numbering) in each of theantibody heavy chains. In a further specific embodiment, said knobmodification comprises the amino acid substitution T366W and the holemodification comprises the amino acid substitutions T366S, L368A andY407V. In still a further specific embodiment, said IFNα2 molecule isfused to the antibody heavy chain comprising the knob modification. Inan even more specific embodiment, said antibody comprises thepolypeptide sequences of SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72,or variants thereof that retain functionality.

In another particular embodiment, the antibody according to theinvention is a full-length human IgG₁ antibody comprising the heavychain variable region sequence of SEQ ID NO: 4 and the light chainvariable region sequence of SEQ ID NO: 2, said antibody comprising inthe Fc region a modification reducing binding affinity of the antibodyto FcγRIIIa and a knob-into-hole modification, said knob-into-holemodification comprising a knob modification in one of the antibody heavychains and a hole modification in the other one of the antibody heavychains, and said antibody having an IFNα2 molecule fused at theN-terminal amino acid to the C-terminal amino acid of one of theantibody heavy chains through a peptide linker. In a specificembodiment, said modification reducing binding affinity of the antibodyto FcγRIIIa comprises the amino acid substitutions L234A, L235A andP329G (EU numbering) in each of the antibody heavy chains. In a furtherspecific embodiment, said knob modification comprises the amino acidsubstitution T366W and the hole modification comprises the amino acidsubstitutions T366S, L368A and Y407V. In still a further specificembodiment, said IFNα2 molecule is fused to the antibody heavy chaincomprising the knob modification. In an even more specific embodiment,said antibody comprises the polypeptide sequences of SEQ ID NO: 50, SEQID NO: 52 and SEQ ID NO: 54, or variants thereof that retainfunctionality.

In still another particular embodiment, the antibody according to theinvention is a full-length human IgG₁ antibody comprising the heavychain variable region sequence of SEQ ID NO: 4 and a light chainvariable region sequence selected from the group of SEQ ID NO: 36, SEQID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46and SEQ ID NO: 48, said antibody comprising in the Fc region amodification reducing binding affinity of the antibody to FcγRIIIa and aknob-into-hole modification, said knob-into-hole modification comprisinga knob modification in one of the antibody heavy chains and a holemodification in the other one of the antibody heavy chains, and saidantibody having an IFNα2 molecule fused at the N-terminal amino acid tothe C-terminal amino acid of one of the antibody heavy chains through apeptide linker. In a specific embodiment, said modification reducingbinding affinity of the antibody to FcγRIIIa comprises the amino acidsubstitutions L234A, L235A and P329G (EU numbering) in each of theantibody heavy chains. In a further specific embodiment, said knobmodification comprises the amino acid substitution T366W and the holemodification comprises the amino acid substitutions T366S, L368A andY407V. In still a further specific embodiment, said IFNα2 molecule isfused to the antibody heavy chain comprising the knob modification. Inan even more specific embodiment, said antibody comprises thepolypeptide sequences of SEQ ID NO: 52, SEQ ID NO: 54 and a polypeptidesequence selected from the group of SEQ ID NO: 96, SEQ ID NO: 98, SEQ IDNO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106 and SEQ ID NO:108, or variants thereof that retain functionality.

In a further embodiment, the antibody of the invention comprises thepolypeptide sequences of SEQ ID NO: 92, SEQ ID NO: 52 and SEQ ID NO: 54,or variants thereof that retain functionality. In another embodiment,the antibody according to the invention comprises the polypeptidesequences of SEQ ID NO: 94, SEQ ID NO: 52 and SEQ ID NO: 54, or variantsthereof that retain functionality. In still a further embodiment, theantibody according to the invention comprises the polypeptide sequencesof SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 60, or variants thereofthat retain functionality. In a further embodiment, the antibodyaccording to the invention comprises the polypeptide sequences of SEQ IDNO: 62, SEQ ID NO: 64 and SEQ ID NO: 66, or variants thereof that retainfunctionality. In a further embodiment, the antibody according to theinvention comprises the polypeptide sequences of SEQ ID NO: 74, SEQ IDNO: 76 and SEQ ID NO: 78, or variants thereof that retain functionality.In a further embodiment, the antibody according to the inventioncomprises the polypeptide sequences of SEQ ID NO: 80, SEQ ID NO: 82 andSEQ ID NO: 84, or variants thereof that retain functionality. In afurther embodiment, the antibody according to the invention comprisesthe polypeptide sequences of SEQ ID NO: 86, SEQ ID NO: 88 and SEQ ID NO:90, or variants thereof that retain functionality.

In an alternative embodiment, the antibody according to the invention isa full-length human IgG₁ antibody lacking one of the two Fab fragmentsand comprising the heavy chain variable region sequence of SEQ ID NO: 16and the light chain variable region sequence of SEQ ID NO: 14, saidantibody comprising in the Fc region a modification reducing bindingaffinity of the antibody to FcγRIIIa and a knob-into-hole modification,said knob-into-hole modification comprising a knob modification in oneof the antibody heavy chains and a hole modification in the other one ofthe antibody heavy chains, and said antibody having an IFNα2 moleculefused at the N-terminal amino acid to the C-terminal amino acid of oneof the antibody heavy chains through a peptide linker. In a specificembodiment, said modification reducing binding affinity of the antibodyto FcγRIIIa comprises the amino acid substitutions L234A, L235A andP329G (EU numbering) in each of the antibody heavy chains. In a furtherspecific embodiment, said knob modification comprises the amino acidsubstitution T366W and the hole modification comprises the amino acidsubstitutions T366S, L368A and Y407V. In still a further specificembodiment, said IFNα2 molecule is fused to the antibody heavy chaincomprising the knob modification. In an even more specific embodiment,said antibody comprises the polypeptide sequences of SEQ ID NO: 68, SEQID NO: 70 and SEQ ID NO: 116, or variants thereof that retainfunctionality.

In another alternative embodiment, the antibody according to theinvention is a full-length human IgG₁ antibody lacking one of the twoFab fragments and comprising the heavy chain variable region sequence ofSEQ ID NO: 4 and the light chain variable region sequence of SEQ ID NO:2, said antibody comprising in the Fc region a modification reducingbinding affinity of the antibody to FcγRIIIa and a knob-into-holemodification, said knob-into-hole modification comprising a knobmodification in one of the antibody heavy chains and a hole modificationin the other one of the antibody heavy chains, and said antibody havingan IFNα2 molecule fused at the N-terminal amino acid to the C-terminalamino acid of one of the antibody heavy chains through a peptide linker.In a specific embodiment, said modification reducing binding affinity ofthe antibody to FcγRIIIa comprises the amino acid substitutions L234A,L235A and P329G (EU numbering) in each of the antibody heavy chains. Ina further specific embodiment, said knob modification comprises theamino acid substitution T366W and the hole modification comprises theamino acid substitutions T366S, L368A and Y407V. In still a furtherspecific embodiment, said IFNα2 molecule is fused to the antibody heavychain comprising the knob modification. In an even more specificembodiment, said antibody comprises the polypeptide sequences of SEQ IDNO: 50, SEQ ID NO: 52 and SEQ ID NO: 116, or variants thereof thatretain functionality.

In still another alternative embodiment, the antibody according to theinvention is a full-length human IgG₁ antibody lacking one of the twoFab fragments and comprising the heavy chain variable region sequence ofSEQ ID NO: 4 and a light chain variable region sequence selected fromthe group of SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48, said antibody comprisingin the Fc region a modification reducing binding affinity of theantibody to FcγRIIIa and a knob-into-hole modification, saidknob-into-hole modification comprising a knob modification in one of theantibody heavy chains and a hole modification in the other one of theantibody heavy chains, and said antibody having an IFNα2 molecule fusedat the N-terminal amino acid to the C-terminal amino acid of one of theantibody heavy chains through a peptide linker. In a specificembodiment, said modification reducing binding affinity of the antibodyto FcγRIIIa comprises the amino acid substitutions L234A, L235A andP329G (EU numbering) in each of the antibody heavy chains. In a furtherspecific embodiment, said knob modification comprises the amino acidsubstitution T366W and the hole modification comprises the amino acidsubstitutions T366S, L368A and Y407V. In still a further specificembodiment, said IFNα2 molecule is fused to the antibody heavy chaincomprising the knob modification. In an even more specific embodiment,said antibody comprises the polypeptide sequences of SEQ ID NO: 52, SEQID NO: 116 and a polypeptide sequence selected from the group of SEQ IDNO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104,SEQ ID NO: 106 and SEQ ID NO: 108, or variants thereof that retainfunctionality.

In a further embodiment, the antibody of the invention comprises thepolypeptide sequences of SEQ ID NO: 92, SEQ ID NO: 52 and SEQ ID NO:116, or variants thereof that retain functionality. In anotherembodiment, the antibody according to the invention comprises thepolypeptide sequences of SEQ ID NO: 94, SEQ ID NO: 52 and SEQ ID NO:116, or variants thereof that retain functionality. In still a furtherembodiment, the antibody according to the invention comprises thepolypeptide sequences of SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO:116, or variants thereof that retain functionality. In a furtherembodiment, the antibody according to the invention comprises thepolypeptide sequences of SEQ ID NO: 62, SEQ ID NO: 64 and SEQ ID NO:116, or variants thereof that retain functionality. In a furtherembodiment, the antibody according to the invention comprises thepolypeptide sequences of SEQ ID NO: 74, SEQ ID NO: 76 and SEQ ID NO:116, or variants thereof that retain functionality. In a furtherembodiment, the antibody according to the invention comprises thepolypeptide sequences of SEQ ID NO: 80, SEQ ID NO: 82 and SEQ ID NO:116, or variants thereof that retain functionality. In a furtherembodiment, the antibody according to the invention comprises thepolypeptide sequences of SEQ ID NO: 86, SEQ ID NO: 88 and SEQ ID NO:116, or variants thereof that retain functionality.

Antibodies of the invention include those that have sequences that areat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the sequences set forth in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108 and 116, including functionalfragments or variants thereof. The invention also encompasses antibodiescomprising these sequences with conservative amino acid substitutions.

Polynucleotides

The invention further provides polynucleotides encoding an antibody asdescribed herein or an antigen binding portion thereof.

Polynucleotides of the invention include those that are at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109 and 117, including functionalfragments or variants thereof.

The polynucleotides encoding antibodies of the invention may beexpressed as a single polynucleotide that encodes the entire antibody oras multiple (e.g., two or more) polynucleotides that are co-expressed.Polypeptides encoded by polynucleotides that are co-expressed mayassociate through, e.g., disulfide bonds or other means to form afunctional antibody. For example, the light chain portion of an antibodymay be encoded by a separate polynucleotide from the heavy chain portionof the antibody. When co-expressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the antibody. Inanother example, the heavy chain portion of the antibody comprising aneffector moiety could be encoded by a separate polynucleotide from theother heavy chain portion of the antibody. When co-expressed, the heavychain polypeptides will associate to form a functional antibody(together with the light chain polypeptide(s)).

In one embodiment, the present invention is directed to a polynucleotideencoding an antibody or an antigen binding portion thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence as shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48. In anotherembodiment, the present invention is directed to a polynucleotideencoding an antibody or an antigen binding portion thereof, wherein thepolynucleotide comprises a sequence that encodes a polypeptide sequenceas shown in SEQ ID NO 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108 or 116. In another embodiment, the invention is further directed toa polynucleotide encoding an antibody or an antigen binding portionthereof, wherein the polynucleotide comprises a sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence shown SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109 or 117. In another embodiment,the invention is directed to a polynucleotide encoding an antibody or anantigen binding portion thereof, wherein the polynucleotide comprises anucleic acid sequence shown in SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 117. In anotherembodiment, the invention is directed to a polynucleotide encoding anantibody or antigen binding portion thereof, wherein the polynucleotidecomprises a sequence that encodes a variable region sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to anamino acid sequence of SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48. In anotherembodiment, the invention is directed to a polynucleotide encoding anantibody or an antigen binding portion thereof, wherein thepolynucleotide comprises a sequence that encodes a polypeptide sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toan amino acid sequence of SEQ ID NO 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108 or 116. The invention encompasses a polynucleotideencoding an antibody or an antigen binding portion thereof, wherein thepolynucleotide comprises a sequence that encodes the variable regionsequences of SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 with conservative aminoacid substitutions. The invention also encompasses a polynucleotideencoding an antibody of the invention or an antigen binding portionthereof, wherein the polynucleotide comprises a sequence that encodesthe polypeptide sequences of SEQ ID NO 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108 or 116 with conservative amino acid substitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

Antibodies of the invention may be obtained, for example, by solid-statepeptide synthesis (e.g. Merrifield solid phase synthesis) or recombinantproduction. For recombinant production one or more polynucleotideencoding the antibody (fragment), e.g., as described above, is isolatedand inserted into one or more vectors for further cloning and/orexpression in a host cell. Such polynucleotide may be readily isolatedand sequenced using conventional procedures. In one embodiment a vector,preferably an expression vector, comprising one or more of thepolynucleotides of the invention is provided. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing the coding sequence of an antibody (fragment) alongwith appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Maniatis et al., MOLECULAR CLONING:A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); andAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing Associates and Wiley Interscience, N.Y (1989). The expressionvector can be part of a plasmid, virus, or may be a nucleic acidfragment. The expression vector includes an expression cassette intowhich the polynucleotide encoding the antibody (fragment) (i.e. thecoding region) is cloned in operable association with a promoter and/orother transcription or translation control elements. As used herein, a“coding region” is a portion of nucleic acid which consists of codonstranslated into amino acids. Although a “stop codon” (TAG, TGA, or TAA)is not translated into an amino acid, it may be considered to be part ofa coding region, if present, but any flanking sequences, for examplepromoters, ribosome binding sites, transcriptional terminators, introns,5′ and 3′ untranslated regions, and the like, are not part of a codingregion. Two or more coding regions can be present in a singlepolynucleotide construct, e.g. on a single vector, or in separatepolynucleotide constructs, e.g. on separate (different) vectors.Furthermore, any vector may contain a single coding region, or maycomprise two or more coding regions, e.g. a vector of the presentinvention may encode one or more polypeptides, which are post- orco-translationally separated into the final proteins via proteolyticcleavage. In addition, a vector, polynucleotide, or nucleic acid of theinvention may encode heterologous coding regions, either fused orunfused to a polynucleotide encoding the antibody (fragment) of theinvention, or variant or derivative thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit â-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the antibody is desired, DNA encoding a signal sequence may be placedupstream of the nucleic acid encoding an antibody of the invention or anantigen binding portion thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase. Exemplary amino acid sequences of secretory signalpeptides are shown in SEQ ID NOs 135-137.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling theantibody may be included within or at the ends of the antibody(fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) an antibody of theinvention. As used herein, the term “host cell” refers to any kind ofcellular system which can be engineered to generate the antibodies ofthe invention or fragments thereof. Host cells suitable for replicatingand for supporting expression of antibodies are well known in the art.Such cells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the antibody for clinical applications.Suitable host cells include prokaryotic microorganisms, such as E. coli,or various eukaryotic cells, such as Chinese hamster ovary cells (CHO),insect cells, or the like. For example, polypeptides may be produced inbacteria in particular when glycosylation is not needed. Afterexpression, the polypeptide may be isolated from the bacterial cellpaste in a soluble fraction and can be further purified. In addition toprokaryotes, eukaryotic microbes such as filamentous fungi or yeast aresuitable cloning or expression hosts for polypeptide-encoding vectors,including fungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr⁻ CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one embodiment, the host cell is a eukaryotic cell,preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell,a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0,Sp20 cell). Standard technologies are known in the art to expressforeign genes in these systems. Cells expressing a polypeptidecomprising either the heavy or the light chain of an antibody, may beengineered so as to also express the other of the antibody chains suchthat the expressed product is an antibody that has both a heavy and alight chain.

In one embodiment, a method of producing an antibody according to theinvention is provided, wherein the method comprises culturing a hostcell comprising a polynucleotide encoding the antibody, as providedherein, under conditions suitable for expression of the antibody, andrecovering the antibody from the host cell (or host cell culturemedium).

Where an antibody is fused to an effector moiety, these components aregenetically fused to each other. Antibodies can be designed such thatits components are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Additional sequences may also be included toincorporate a cleavage site to separate the individual components of thefusion if desired, for example an endopeptidase recognition sequence.

In certain embodiments the antibodies of the invention comprise at leastan antibody variable region capable of binding to ASGPR. Variableregions can form part of and be derived from naturally or non-naturallyoccurring antibodies and fragments thereof. Methods to producepolyclonal antibodies and monoclonal antibodies are well known in theart (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”, ColdSpring Harbor Laboratory, 1988). Non-naturally occurring antibodies canbe constructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see e.g. U.S. Pat. No.5,969,108 to McCafferty).

Any animal species of antibody can be used in the invention.Non-limiting antibodies useful in the present invention can be ofmurine, primate, or human origin. If the antibody is intended for humanuse, a chimeric form of antibody may be used wherein the constantregions of the antibody are from a human. A humanized or fully humanform of the antibody can also be prepared in accordance with methodswell known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter).Humanization may be achieved by various methods including, but notlimited to (a) grafting the non-human (e.g., donor antibody) CDRs ontohuman (e.g. recipient antibody) framework and constant regions with orwithout retention of critical framework residues (e.g. those that areimportant for retaining good antigen binding affinity or antibodyfunctions), (b) grafting only the non-human specificity-determiningregions (SDRs or a-CDRs; the residues critical for the antibody-antigeninteraction) onto human framework and constant regions, or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Humanizedantibodies and methods of making them are reviewed, e.g., in Almagro andFransson, Front Biosci 13, 1619-1633 (2008), and are further described,e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al.,Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525(1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984);Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994);Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR)grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimkaet al., Br J Cancer 83, 252-260 (2000) (describing the “guidedselection” approach to FR shuffling). Particular antibodies according tothe invention are human antibodies. Human antibodies and human variableregions can be produced using various techniques known in the art. Humanantibodies are described generally in van Dijk and van de Winkel, CurrOpin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20,450-459 (2008). Human variable regions can form part of and be derivedfrom human monoclonal antibodies made by the hybridoma method (see e.g.Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)). Human antibodies and humanvariable regions may also be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125(2005). Human antibodies and human variable regions may also begenerated by isolating Fv clone variable region sequences selected fromhuman-derived phage display libraries (see e.g., Hoogenboom et al. inMethods in Molecular Biology 178, 1-37 (O'Brien et al., ed., HumanPress, Totowa, N.J., 2001); and McCafferty et al., Nature 348, 552-554;Clackson et al., Nature 352, 624-628 (1991)). Phage typically displayantibody fragments, either as single-chain Fv (scFv) fragments or as Fabfragments. A detailed description of the preparation of antibodies byphage display can be found in the Examples.

In certain embodiments, the antibodies of the present invention areengineered to have enhanced binding affinity according to, for example,the methods disclosed in PCT publication WO 2011/020783 (see Examplesrelating to affinity maturation) or U.S. Pat. Appl. Publ. No.2004/0132066, the entire contents of which are hereby incorporated byreference. The ability of the antibody of the invention to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional bindingassays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays maybe used to identify an antibody that competes with a reference antibodyfor binding to a particular antigen, e.g. an antibody that competes withthe 51A12 antibody for binding to ASGPR. In certain embodiments, such acompeting antibody binds to the same epitope (e.g. a linear or aconformational epitope) that is bound by the reference antibody.Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris (1996) “Epitope Mapping Protocols”, inMethods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In anexemplary competition assay, immobilized antigen (e.g. ASGPR) isincubated in a solution comprising a first labeled antibody that bindsto the antigen (e.g. 51A12 antibody) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to the antigen. The second antibody may be present in ahybridoma supernatant. As a control, immobilized antigen is incubated ina solution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to the antigen, excess unbound antibody isremoved, and the amount of label associated with immobilized antigen ismeasured. If the amount of label associated with immobilized antigen issubstantially reduced in the test sample relative to the control sample,then that indicates that the second antibody is competing with the firstantibody for binding to the antigen. See Harlow and Lane (1988)Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

Antibodies prepared as described herein may be purified by art-knowntechniques such as high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, affinity chromatography, sizeexclusion chromatography, and the like. The actual conditions used topurify a particular protein will depend, in part, on factors such as netcharge, hydrophobicity, hydrophilicity etc., and will be apparent tothose having skill in the art. For affinity chromatography purificationan antibody, ligand, receptor or antigen can be used to which theantibody binds. For example, for affinity chromatography purification ofantibodies of the invention, a matrix with protein A or protein G may beused. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate an antibody essentiallyas described in the Examples. The purity of the antibody can bedetermined by any of a variety of well known analytical methodsincluding gel electrophoresis, high pressure liquid chromatography, andthe like. For example, the heavy chain fusion proteins expressed asdescribed in the Examples were shown to be intact and properly assembledas demonstrated by reducing SDS-PAGE (see e.g. FIG. 13-19).

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the antibodies provided herein, e.g., for use in anyof the below therapeutic methods. In one embodiment, a pharmaceuticalcomposition comprises any of the antibodies provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises any of the antibodies providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Further provided is a method of producing an antibody of the inventionin a form suitable for administration in vivo, the method comprising (a)obtaining an antibody according to the invention, and (b) formulatingthe antibody with at least one pharmaceutically acceptable carrier,whereby a preparation of antibody is formulated for administration invivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more antibody dissolved ordispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecularentities and compositions that are generally non-toxic to recipients atthe dosages and concentrations employed, i.e. do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one antibody andoptionally an additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards or correspondingauthorities in other countries. Preferred compositions are lyophilizedformulations or aqueous solutions. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, buffers, dispersionmedia, coatings, surfactants, antioxidants, preservatives (e.g.antibacterial agents, antifungal agents), isotonic agents, absorptiondelaying agents, salts, preservatives, antioxidants, proteins, drugs,drug stabilizers, polymers, gels, binders, excipients, disintegrationagents, lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. Antibodies of the present invention (and any additionaltherapeutic agent) can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrasplenically, intrarenally,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, by inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g. liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference). Parenteraladministration, in particular intravenous injection, is most commonlyused for administering polypeptide molecules such as the antibodies ofthe invention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the antibodies of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the antibodies maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. Sterile injectable solutions areprepared by incorporating the antibodies of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated below, as required. Sterility may be readilyaccomplished, e.g., by filtration through sterile filtration membranes.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and/or the other ingredients. In the case ofsterile powders for the preparation of sterile injectable solutions,suspensions or emulsion, the preferred methods of preparation arevacuum-drying or freeze-drying techniques which yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the antibodies mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theantibodies may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Pharmaceutical compositions comprising the antibodies of the inventionmay be manufactured by means of conventional mixing, dissolving,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions may be formulated in conventional mannerusing one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the proteinsinto preparations that can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

The antibodies may be formulated into a composition in a free acid orbase, neutral or salt form. Pharmaceutically acceptable salts are saltsthat substantially retain the biological activity of the free acid orbase. These include the acid addition salts, e.g., those formed with thefree amino groups of a proteinaceous composition, or which are formedwith inorganic acids such as for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric or mandelicacid. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine. Pharmaceutical salts tend to bemore soluble in aqueous and other protic solvents than are thecorresponding free base forms.

Therapeutic Methods and Compositions

Any of the antibodies provided herein may be used in therapeuticmethods.

For use in therapeutic methods, antibodies of the invention would beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners.

In one aspect, antibodies of the invention for use as a medicament areprovided. In further aspects, antibodies of the invention for use intreating a disease are provided. In certain embodiments, antibodies ofthe invention for use in a method of treatment are provided. In oneembodiment, the invention provides an antibody as described herein foruse in the treatment of a disease in an individual in need thereof. Incertain embodiments, the invention provides an antibody for use in amethod of treating an individual having a disease comprisingadministering to the individual a therapeutically effective amount ofthe antibody. In certain embodiments the disease to be treated is aliver disease. Exemplary liver diseases include hepatits, cirrhosis, orliver cancer such as hepatocellular carcinoma. In a particularembodiment the disease is a viral infection, particularly a hepatitisvirus infection, more particularly HBV infection. In another particularembodiment the disease is cancer, particularly liver cancer, moreparticularly hepatocellular carcinoma (HCC). In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-viral agent if the disease to be treated is a viralinfection or an anti-cancer agent if the disease to be treated iscancer. An “individual” according to any of the above embodiments is amammal, preferably a human.

In a further aspect, the invention provides for the use of an antibodyof the invention in the manufacture or preparation of a medicament forthe treatment of a disease in an individual in need thereof. In oneembodiment, the medicament is for use in a method of treating a diseasecomprising administering to an individual having the disease atherapeutically effective amount of the medicament. In certainembodiments the disease to be treated is a liver disease. In aparticular embodiment the disease is a viral infection, particularly ahepatitis virus infection, more particularly HBV infection. In otherembodiments the disease to be treated is cancer. In a particularembodiment the disease is liver cancer, particularly hepatocellularcarcinoma (HCC). In one embodiment, the method further comprisesadministering to the individual a therapeutically effective amount of atleast one additional therapeutic agent, e.g., an anti-viral agent if thedisease to be treated is a viral infection or an anti-cancer agent ifthe disease to be treated is cancer. An “individual” according to any ofthe above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of an antibody of the invention. In oneembodiment a composition is administered to said individual, comprisingantibody of the invention in a pharmaceutically acceptable form. Incertain embodiments the disease to be treated is a liver disease. In aparticular embodiment the disease is a viral infection, particularly ahepatitis virus infection, more particularly HBV infection. In otherembodiments the disease to be treated is cancer. In a particularembodiment, the disease is liver cancer, particularly hepatocellularcarcinoma (HCC). In certain embodiments the method further comprisesadministering to the individual a therapeutically effective amount of atleast one additional therapeutic agent, e.g., an anti-viral agent if thedisease to be treated is a viral infection or an anti-cancer agent ifthe disease to be treated is cancer. An “individual” according to any ofthe above embodiments may be a mammal, preferably a human.

The antibodies of the invention are also useful as diagnostic reagents.The binding of an antibody to an antigenic determinant can be readilydetected by a label attached to the antibody or by using a labeledsecondary antibody specific for the antibody of the invention.

In some embodiments, an effective amount of an antibody of the inventionis administered to a cell. In other embodiments, a therapeuticallyeffective amount of an antibody of the invention is administered to anindividual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the route of administration, the body weight ofthe patient, the type of antibody, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous or concurrent therapeutic interventions,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The practitioner responsible foradministration will, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s) for theindividual subject. Various dosing schedules including but not limitedto single or multiple administrations over various time-points, bolusadministration, and pulse infusion are contemplated herein.

The antibody is suitably administered to the patient at one time or overa series of treatments. Depending on the type and severity of thedisease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibodycan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. One typical daily dosage might range from about 1μg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment would generally be sustained until adesired suppression of disease symptoms occurs. One exemplary dosage ofthe antibody would be in the range from about 0.005 mg/kg to about 10mg/kg. In other non-limiting examples, a dose may also comprise fromabout 1 μg/kg body weight, about 5 μg/kg body weight, about 10 μg/kgbody weight, about 50 μg/kg body weight, about 100 μg/kg body weight,about 200 μg/kg body weight, about 350 μg/kg body weight, about 500μg/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body weight,about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kgbody weight, about 200 mg/kg body weight, about 350 mg/kg body weight,about 500 mg/kg body weight, to about 1000 mg/kg body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 5 mg/kg body weight to about 100 mg/kg body weight, about 5 μg/kgbody weight to about 500 mg/kg body weight etc., can be administered,based on the numbers described above. Thus, one or more doses of about0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof)may be administered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the antibody). An initial higher loading dose, followed by one ormore lower doses may be administered. However, other dosage regimens maybe useful. The progress of this therapy is easily monitored byconventional techniques and assays.

The antibodies of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the antibodies of the invention, or pharmaceuticalcompositions thereof, are administered or applied in a therapeuticallyeffective amount. Determination of a therapeutically effective amount iswell within the capabilities of those skilled in the art, especially inlight of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the antibodies which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the antibodies may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the antibodies described herein willgenerally provide therapeutic benefit without causing substantialtoxicity. Toxicity and therapeutic efficacy of an antibodies can bedetermined by standard pharmaceutical procedures in cell culture orexperimental animals. Cell culture assays and animal studies can be usedto determine the LD₅₀ (the dose lethal to 50% of a population) and theED₅₀ (the dose therapeutically effective in 50% of a population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which can be expressed as the ratio LD₅₀/ED₅₀. Antibodies thatexhibit large therapeutic indices are preferred. In one embodiment, theantibody according to the present invention exhibits a high therapeuticindex. The data obtained from cell culture assays and animal studies canbe used in formulating a range of dosages suitable for use in humans.The dosage lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon a variety of factors, e.g., the dosageform employed, the route of administration utilized, the condition ofthe subject, and the like. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition (see, e.g., Fingl et al., 1975, in: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated hereinby reference in its entirety).

The attending physician for patients treated with antibodies of theinvention would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The antibodies of the invention may be administered in combination withone or more other agents in therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. The term “therapeutic agent” encompasses any agentadministered to treat a symptom or disease in an individual in need ofsuch treatment. Such additional therapeutic agent may comprise anyactive ingredients suitable for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. In certain embodiments, an additional therapeuticagent is an anti-viral agent. In other embodiments, an additionaltherapeutic agent is an anti-cancer agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of antibody used, the type ofdisorder or treatment, and other factors discussed above. The antibodiesare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther therapeutic agent. The article of manufacture in this embodimentof the invention may further comprise a package insert indicating thatthe compositions can be used to treat a particular condition.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al, Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory press, Cold spring Harbor, New York, 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in: Kabat, E. A. et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIHPublication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing

Gene Synthesis

Desired gene segments, where required, were either generated by PCRusing appropriate templates or were synthesized at Geneart AG(Regensburg, Germany) from synthetic oligonucleotides and PCR productsby automated gene synthesis. In cases where no exact gene sequence wasavailable, oligonucleotide primers were designed based on sequences fromclosest homologues and the genes were isolated by RT-PCR from RNAoriginating from the appropriate tissue. The gene segments flanked bysingular restriction endonuclease cleavage sites were cloned intostandard cloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow subcloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells. SEQ IDNOs 135-137 give exemplary leader peptides.

Cloning of Antigen Expression Vectors

The amplified DNA fragments encoding the antigen of interest wereinserted in frame into a mammalian recipient vector downstream of ahuman IgG₁ Fc coding fragment serving as solubility- and purificationtag (FIG. 1). Expression of antigen-Fc fusions with a wild type Fcsequence (SEQ ID NOs 123, 125, 127, 129, 131, 133) resulted inhomodimeric molecules (avi-Fc-human ASGPR H1 stalk: SEQ ID NO: 124,avi-Fc-cynomolgus ASGPR H1 stalk: SEQ ID NO: 126, avi-Fc-human ASGPR H1stalk CRD: SEQ ID NO: 130, avi-Fc-cynomolgus ASGPR H1 stalk CRD: SEQ IDNO: 132). Protein CLEC10A was identified as the closest homologue toASGPR H1 and the constructs avi-Fc-human CLEC10A stalk (SEQ ID NO: 128)and avi-Fc-human CLEC10A stalk CRD (SEQ ID NO: 134) were expressed fortesting the specificity of the selected binders. In order to express theantigen in a monomeric state, the DNA fragment was fused to an Fc partcontaining the “hole” mutations (SEQ ID NOs 117, 119) and wasco-expressed with an Fc-“knob” (SEQ ID NO: 121) counterpart (Fc-humanASGPR H1 CRD: SEQ ID NOs 118 and 122, avi-Fc-human CLEC10A CRD: SEQ IDNOs 120 and 122). The antigen expression was generally driven by an MPSVpromoter and transcription was terminated by a synthetic polyA signalsequence located downstream of the CDS. In addition, all constructscontained an N-terminal Avi tag allowing specific biotinylation duringco-expression with Bir A biotin ligase. In addition to the expressioncassette, each vector contained an EBV oriP sequence for autonomousreplication in EBV-EBNA expressing cell lines.

Production and Purification of Antigens and Antibodies

Both antigens and antibodies were transiently transfected into HEK 293cells, stably expressing the EBV-derived protein EBNA. A simultaneouslyco-transfected plasmid encoding biotin ligase Bir A allowed Avitag-specific biotinylation in vivo. The proteins were then purifiedusing a protein A column followed by gel filtration.

Generation of a Generic Lambda Fab-Library

A generic lambda antibody library in the Fab-format was generated on thebasis of human germline genes using the following V-domain pairings:V13_(—)19 lambda light chain with VH3_(—)23 heavy chain resulting in aDP47-lambda library. The library was randomized in CDR3 of the lightchain (L3) and CDR3 of the heavy chain (H3) and was assembled from threefragments by “splicing by overlapping extension” (SOE) PCR. Fragment 1comprises the 5′ end of the antibody gene including randomized L3,fragment 2 is a central constant fragment spanning from the end of L3 tothe beginning of H3, whereas fragment 3 comprises randomized H3 and the3′ portion of the Fab fragment. The following primer combinations wereused to generate library fragments for library: fragment 1 (LMB3 (SEQ IDNO: 146)—V1_(—)3_(—)19 L3r primers (SEQ ID NOs 143-145)), fragment 2(RJH80 (SEQ ID NO: 148)—DP47CDR3_ba (mod) (SEQ ID NO: 149)), fragment 3(DP47-v4 primers (SEQ ID NO: 140-142)—fdseqlong (SEQ ID NO: 147) (Table1). PCR parameters for production of library fragments were 5 mininitial denaturation at 94° C., 25 cycles of 60 sec 94° C., 60 sec 55°C., 60 sec 72° C. and terminal elongation for 10 min at 72° C. Forassembly PCR, using equimolar ratios of the 3 fragments as template,parameters were 3 min initial denaturation at 94° C. and 5 cycles of 60s 94° C., 60 sec 55° C., 120 sec 72° C. At this stage, outer primerswere added and additional 20 cycles were performed prior to a terminalelongation for 10 min at 72° C. (FIG. 2). After assembly of sufficientamounts of full length randomized Fab fragments, they were digested withNcoI/NheI alongside with similarly treated acceptor phagemid vector. 15μg of Fab library insert were ligated with 13.3 μg of phagemid vector.Purified ligations were used for 60 transformations resulting in 1.5×10⁹transformants. Phagemid particles displaying the Fab library wererescued and purified by PEG/NaCl purification to be used for selections.

TABLE 1Sequences of primers used for the generation of the generic lambda library.SEQ ID NO NAME SEQUENCE 140 DP47-v4-4CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-3-4-GAC-TAC- TGGGGCCAAGGAACCCTGGTCACCGTCTCG 1 : G/D = 20, E/V/S =10, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15, A/D/T/R/P/L/V/N/W/F/I/E =4.6%; 3: G/A/Y = 20, P/W/S/D/T = 8%; 4: F = 46, L/M = 15, G/I/Y =8%; 5: K = 70, R = 30% 141 DP47-v4-6CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-3-4-GAC-TAC- TGGGGCCAAGGAACCCTGGTCACCGTCTCG 1: G/D = 20, E/V/S =10, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15, A/D/T/R/P/L/V/N/W/F/I/E =4.6%; 3: G/A/Y = 20, P/W/S/D/T = 8%; 4: F = 46, L/M = 15, G/I/Y =8%; 5: K = 70, R = 30% 142 DP47-v4-8CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-2-2-3-4-GAC-TAC- TGGGGCCAAGGAACCCTGGTCACCGTCTCG 1: G/D = 20, E/V/S =10, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15, A/D/T/R/P/L/V/N/W/F/I/E =4,6%; 3 : G/A/Y = 20, P/W/S/D/T = 8%; 4: F = 46, L/M = 15, G/I/Y =8%; 5: K = 70, R = 30% 143 V1_3_19_L3r_VGGACGGTCAGCTTGGTCCCTCCGCCGAATAC V

 A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGCunderlined: 60% original base and 40% randomization as Mbolded and italic: 60% original base and 40% randomization as N 144V1_3_19_L3r_HV GGACGGTCAGCTTGGTCCCTCCGCCGAATAC C

 A

 A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGCunderlined: 60% original base and 40% randomization as Mbolded and italic: 60% original base and 40% randomization as N 145V1_3_19_L3r_HLV GGACGGTCAGCTTGGTCCCTCCGCCGAATAC R

 V

 A

 A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTC CGCunderlined: 60% original base and 40% randomization as Mbolded and italic: 60% original base and 40% randomization as N 146 LMB3CAGGAAACAGCTATGACCATGATTAC 147 fdseqlong GACGTTAGTAAATGAATTTTCTGTATGAGG148 RJH80 TTCGGCGGAGGGACCAAGCTGACCGTCC 149 DP47CDR3_baCGCACAGTAATATACGGCCGTGTCC (mod)

Selection of Anti-Human ASGPR H1 Binders from a Generic Lambda FabLibrary

Selections against the complete or fragments of the extracellular domain(ECD) of human ASGPR H1 were carried out using HEK293-expressedmonomeric or dimeric human ASGPR protein fragments fused to theFc-portion of a human IgG1 antibody (SEQ ID NO: 118, 120, 124, 126, 128,130, 132, 134). While ASGPR H1 CRD and CLEC10A CRD were expressed asmonomeric Fc fusions using the Fc “knob-into-hole” format (only one Fccarrying a C-terminally fused CRD), all stalk fragments and total ECDswere expressed as homodimeric Fc fusion proteins (FIG. 1). The antigenswere enzymatically biotinylated by co-expression of the biotin ligaseBir A via an N-terminal avi-tag. Panning rounds were performed insolution according to the following pattern: (1) Preclearing of ˜10¹²phagemid particles using human IgG₁ coated at 10 μg/ml onto NUNCmaxisorp plates to avoid Fc-binders, (2) binding of non-Fc bindingphagemid particles from the supernatant of the pre-clearing reaction to100 nM biotinylated antigen protein for 0.5 h in a total volume of 1 ml,(3) capture of biotinylated antigen and attached specifically bindingphage by addition of 5.4×10⁷ streptavidin-coated magnetic beads for 10min, (4) washing of beads using 5×1 ml PBS/Tween-20 and 5×1 ml PBS, (5)elution of phage particles by addition of 1 ml 100 mM triethylamine(TEA) for 10 min and neutralization by addition of 500 μl 1M Tris/HCl pH7.4, (6) Re-infection of log-phase E. coli TG1 cells with the phageparticles in the supernatant, infection with helperphage VCSM13 andsubsequent PEG/NaCl precipitation of phagemid particles to be used insubsequent selection rounds. Selections were carried out over 3-5 roundsusing either constant or decreasing (from 10⁻⁷ M to 2×10⁻⁹ M) antigenconcentrations. In round 2, capture of antigen-phage complexes wasperformed using neutravidin plates instead of streptavidin beads.Specific binders were identified by ELISA as follows: 100 μl of 50 nMbiotinylated human Fc-stalk-CRD, Fc-CRD, or Fc-stalk per well werecoated on neutravidin plates. Fab-containing bacterial supernatants wereadded and binding Fabs were detected via their Flag-tags by using ananti-Flag/HRP secondary antibody.

Clones exhibiting significant signals over background were short-listedfor sequencing (SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25and 27) and further analyses.

Purification of Fabs

Fabs from bacterial cultures (protein sequence of variable domainslisted as SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and28) were purified for the exact analysis of the kinetic parameters. Foreach clone, a 500 ml culture was inoculated with bacteria harboring thecorresponding phagemid and induced with 1 mM IPTG at an OD₆₀₀ 0.9.Afterwards, the cultures were incubated at 25° C. overnight andharvested by centrifugation. After the incubation of the resuspendedpellet for 20 min in 25 ml PPB buffer (30 mM Tris-HCl pH 8, 1 mM EDTA,20% sucrose), bacteria were centrifuged again and the supernatant washarvested. This incubation step was repeated once with 25 ml of a 5 mMMgSO₄ solution. The supernatants of both incubation steps were pooled,filtered and loaded on an IMAC column (His gravitrap, GE Healthcare).Subsequently, the column was washed with 40 ml washing buffer (500 mMNaCl, 20 mM imidazole, 20 mM NaH₂PO₄ pH 7.4). After the elution (500 mMNaCl, 500 mM imidazole, 20 mM NaH₂PO₄ pH 7.4) the eluate was re-bufferedusing PD10 columns (GE Healthcare). The kinetic parameters of thepurified Fabs were then studied by SPR-analysis (Proteon XPR36, Biorad)in a dilution row that ranged from 200 nM to 6.25 nM.

Affinity-Determination by SPR

Affinity (K_(D)) of selected Fab clones was measured by surface plasmonresonance using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated mono- (avi-Fc-human ASGPR H1 CRD, SEQ ID NO: 118) orbivalent (avi-Fc-human ASGPR H1 stalk-CRD, SEQ ID NO: 130) ASGPR H1antigens immobilized on NLC chips by neutravidin capture. Immobilizationof recombinant antigens (ligand): Antigens were diluted with PBST (10 mMphosphate, 150 mM NaCl pH 7.4, 0.005% Tween-20) to 10 μg/ml, theninjected at 30 μl/min at varying contact times, to achieveimmobilization levels of 200, 400 or 800 response units (RU) in verticalorientation. Injection of analytes: For one-shot kinetics measurements,injection direction was changed to horizontal orientation, two-folddilution series of purified Fab (varying concentration ranges between100 and 6.25 nM) were injected simultaneously at 50, 60 or 100 μl/minalong separate channels 1-5, with association times of 150 or 200 s, anddissociation times of 240 or 600 s. Buffer (PBST) was injected along thesixth channel to provide an “in-line” blank for referencing. Associationrate constants (k_(on)) and dissociation rate constants (k_(off)) werecalculated using a simple one-to-one Langmuir binding model in ProteOnManager v3.1 software by simultaneously fitting the association anddissociation sensorgrams. The equilibrium dissociation constant (K_(D))was calculated as the ratio k_(off)/k_(on). Regeneration was performedin horizontal orientation using 10 mM glycine, pH 1.5 at a flow rate of100 μl/min for a contact time of 30 s. Two clones, 51A12 (SEQ ID NO: 002and 004) and 52C4 (SEQ ID NO: 006 and 008), were found to be specific tothe ASGPR H1 CRD. Remarkably, clone 51A12 revealed an affinity in thesubnanomolar range. Clones 5A4 (SEQ ID NO: 010 and 012), 4F3 (SEQ ID NO:014 and 016), R5C2 (SEQ ID NO: 018 and 020), R9E10 (SEQ ID NO: 022 and024), and R9E10 (SEQ ID NO: 026 and 028) were raised either against thestalk region of ASGPR H1 or the interface between the stalk and CRD. Theaffinity to their corresponding human and cynomolgus epitopes wassimilar. In contrast, no binding to avi-Fc-human CLEC10A stalk CRD (SEQID NO: 134) was detected, demonstrating the high specificity of thesebinders. Interestingly, clone 5A4 demonstrated strong binding to thestalk antigen but not to stalk-CRD. The kinetic and thermodynamic dataof all measurements are summarized in Table 2.

TABLE 2 Kinetic and thermodynamic parameters of anti-ASGPR H1 Fabs.huASGPR cyASGPR huASGPR cyASGPR H1 stalk H1 stalk hu/cyASGPR huCLEC10AH1 stalk H1 stalk CRD CRD H1 CRD stalk CRD ka (1/Ms) ka (1/Ms) ka (1/Ms)ka (1/Ms) ka (1/Ms) ka (1/Ms) Antibody kd (1/s) kd (1/s) kd (1/s) kd(1/s) kd (1/s) kd (1/s) clone KD (M) KD (M) KD (M) KD (M) KD (M) KD (M)52C4 1.39 × 10⁵ no binding 3.96 × 10⁻³ 2.86 × 10⁻⁸ 51A12 1.10 × 10⁵ nobinding 6.28 × 10⁻⁵ 5.70 × 10⁻¹⁰ 5A4 2.44 × 10⁵ 3.72 × 10⁵ no binding1.05 × 10⁻³ 1.40 × 10⁻³ 4.30 × 10⁻⁹ 3.76 × 10⁻⁹ 4F3 1.51 × 10⁵ 1.43 ×10⁵ 2.24 × 10⁵ 1.69 × 10⁵ no binding 4.45 × 10⁻³ 4.80 × 10⁻³ 4.08 × 10⁻³3.65 × 10⁻³ 2.95 × 10⁻⁸ 3.36 × 10⁻⁸ 1.82 × 10⁻⁸ 2.16 × 10⁻⁸ R9E10 5.86 ×10⁵ 4.60 × 10⁵ no binding 2.11 × 10⁻³ 1.99 × 10⁻³ 3.60 × 10⁻⁹ 4.34 ×10⁻⁹ R7E12 2.94 × 10⁵ 2.47 × 10⁵ no binding 2.68 × 10⁻³ 2.36 × 10⁻³ 9.12× 10⁻⁹ 9.55 × 10⁻⁹ R5C2 4.23 × 10⁵ 3.34 × 10⁵ no binding 1.12 × 10⁻³1.14 × 10⁻³ 2.65 × 10⁻⁹ 3.41 × 10⁻⁹

Cloning of Variable Antibody Domains into Expression Vectors

All Fabs demonstrating specific binding to their corresponding antigenby SPR were converted into an IgG₁/lambda antibody. Therefor, thePCR-amplified DNA fragments of heavy and light chain v-domains wereinserted in frame into either the human IgG₁ constant heavy chain or thehuman constant lambda light chain containing respective recipientmammalian expression vector. The antibody expression was driven by anMPSV promoter and transcription was terminated by a synthetic polyAsignal sequence located downstream of the CDS. In addition to theexpression cassette each vector contained an EBV oriP sequence forautonomous replication in EBV-EBNA expressing cell lines.

Binding Analysis of the Antibodies to HepG2 Cells

Binding of human IgG₁ anti-ASGPR antibodies to the hepatocellularcarcinoma cell line HepG2 was measured by FACS. Briefly, 0.2 mio cellsper well in a 96 well round bottom plate were incubated in 300 μl withthe anti-ASGPR antibodies at a concentration of 30 μg/ml for 30 min at4° C. Unbound antibody was removed by washing the cells with PBScontaining 0.1% BSA. Bound antibodies were detected with FITC-conjugatedAffiniPure goat anti-human IgG Fc gamma fragment-specific secondaryF(ab′)2 fragment (Jackson ImmunoResearch #109-096-098; working solution1:20 in PBS, 0.1% BSA). After 30 min incubation at 4° C. unboundantibody was removed by washing and cells were fixed using 1% PFA. Cellswere analyzed using BD FACS Cantoll (Software BD DIVA) (FIG. 3). Allantibodies showed strong binding to the HepG2 cells.

Fluorescence Resonance Energy Transfer Assay

The avidity of the IgGs to their epitope on ASGPR-expressing cells wasdetermined by Fluorescence Resonance Energy Transfer (FRET) analysis.For this analysis, the DNA sequence encoding for the SNAP Tag (plasmidpurchased from Cisbio) was amplified by PCR and ligated into anexpression vector, containing the full length human ASGPR H1 sequence(Origene). The resulting fusion protein was comprised of full-lengthASGPR H1 with a C-terminal SNAP tag. HEK293 cells were transfected with10 μg DNA using Lipofectamine 2000 as transfection reagent. After anincubation time of 20 h, cells were washed with PBS and incubated for 1h at 37° C. in LabMed buffer (Cisbio) containing 100 nM SNAP-Lumi4Tb(Cibsio), leading to specific labeling of the SNAP Tag. Subsequently,cells were washed 4 times with LabMed buffer to remove unbound dye. Thelabeling efficiency was determined by measuring the emission of terbiumat 615 nm compared to buffer. Cells were then stored frozen at −80° C.for up to 6 months.

Avidity was measured by adding ASGPR-specific antibodies at aconcentration ranging from 50-0.39 nM to labeled cells (100 cells perwell) followed by addition of anti-humanFc-d2 (final 200 nM per well) asacceptor molecule for the FRET. After an incubation time of 3 h at RTthe emission of the acceptor dye (665 nm) as well as of the donor dye(615 nm) was determined using a fluorescence Reader (Victor 3, PerkinElmer). The ratio of acceptor to donor emission was calculated and theratio of the background control (cells with anti-huFc-d2) subtracted.Curves were analysed in GraphPad Prism5 and K_(D) values calculated(FIG. 4). While clone 4F3 shows the lowest affinity to ASGPR H1stalk-CRD measured by SPR (Table 2), binding intensity as an IgG to thecell surface is driven by strong avidity making 4F3 to the clone withthe strongest binding intensity at low concentrations. In contrast,clone 51A12 which binds to the CRD shows a significantly weaker bindingintensity at low antibody concentrations to cells than to the purifiedantigen in SPR studies.

Binding Competition with a Natural ASGPR Ligand

Competition of the ASGPR antibodies with a desialylated glycoproteinsuch as asialofetuin as a natural ligand for ASGPR was analyzed usingthe hepatocellular carcinoma cell line HepG2. 0.2 mio cells per well ina 96 well round bottom plate were incubated with 40 μl of Alexa488labeled asialofetuin (from fetal calf serum, Sigma Aldrich #A4781, finalconcentration 100 μg/ml) at 4° C. for 30 min. The binding was performedin the presence of calcium, as ligand binding to ASGPR is calciumdependent. Unbound protein was removed by washing the cells once withHBSS containing 0.1% BSA. Then 40 μl of the anti-ASGPR antibodies (30,6, and 1.25 μg/ml final concentration) were added to the cells in thepresence of 100 μg/ml asialofetuin. Cells were incubated for 30 min at4° C. and unbound protein was removed by washing the cells once. AnAPC-conjugated AffiniPure goat anti-human IgG Fc gamma fragment-specificsecondary F(ab′)2 fragment (Jackson Immuno Research #109-136-170;working solution 1:50 in HBSS containing 0.1% BSA) was used as ansecondary antibody. After 30 min incubation at 4° C. unbound secondaryantibody was removed by washing. Cells were fixed using 1% PFA andanalyzed using BD FACS Cantoll (Software BD DIVA). Analysis of both theCRD-specific and the stalk-CRD-specific antibodies revealed thatantibodies bind to ASGPR H1 independently of the presence of theasialofetuin and vice versa, and no binding competition takes place.(FIGS. 5 and 6).

Internalization Study

Uptake of desialylated glycoproteins into liver cells after binding toASGPR is known to occur very rapidly. During this receptor-mediatedendocytosis, the lumen of the endosome becomes acidic allowing thereceptor-ligand complexes to dissociate. While the ligand is targetedfor degradation in lysosomes, ASGPR was shown to recycle back to thecell surface. In order to analyze the retention time of the antibodieson the cell surface, internalization of the ASGPR-antibody complex wasanalyzed using the hepatocellular carcinoma cell line HepG2.ASGPR-positive HepG2 cells were shifted in cell culture medium to 4° C.in order to inhibit internalization. After 45 min incubation with theantibodies (30 μg/ml) on a shaker at 4° C., unbound antibodies wereremoved by washing twice with cold PBS and cells were re-suspended andcultured in pre-warmed medium at 37° C. to re-activate the cellularmetabolism including receptor-mediated endocytosis. One aliquot wastaken immediately and stored on ice which represents time point zero.Remaining cells were incubated at 37° C. and after 5, 15, 30 and 120 minadditional samples were taken and washed with cold PBS to stop furtherinternalization. Cell surface-bound antibodies were detected usingPE-conjugated AffiniPure goat anti-human IgG Fc gamma-specific secondaryF(ab′)2 antibody Fragment (Jackson Immuno Research #109-116-170, workingsolution 1:50). After 30 min incubation at 4° C. unbound antibody wasremoved by washing with PBS containing 0.1% BSA. Cells were fixed using1% PFA and analyzed using BD FACS Cantoll (Software BD DIVA). FIG. 7Ashows exemplary cell surface exposed antibody levels of clones 4F3 and51A12. Interestingly, extracellular antibody signal decreasedsignificantly (up to 60% signal decrease) during the first 30 min butthen decrease delayed for the rest of the time course. This resultindicates that antibodies are internalized very efficiently but theneventually recycle back to the cell surface leading to a dynamic steadystate condition of constant internalization and recycling. In order tosupport this hypothesis, the same experiment was performed, butincubation of the cells with the antibodies was performed in cellculture medium for 45 min at 37° C. These conditions allowreceptor-antibody complexes to be formed and be internalized during theentire incubation time, eventually leading to a steady state of constantendocytosis and recycling. Afterwards, unbound antibody was removed bywashing twice with warm PBS and cells were re-suspended in warm medium.One sample was taken immediately and stored on ice which represents timepoint zero. Remaining cells were incubated at 37° C. and after 5, 15, 30and 120 min additional samples were taken and washed with cold PBS tostop further internalization. Detection of surface-exposed antibodieswas performed as described above. FACS analysis revealed that thedecrease of the signal intensity was less pronounced during the timecourse of the experiment after antibody incubation at 37° C. than 4° C.suggesting that incubation of the antibodies at 37° C. yields in anequilibrium of internalization and recycling of the antibody-receptorcomplex (FIG. 7B). In order to further endorse the hypothesis ofconstant internalization and recycling, internalization of ASGPRH1-specific antibodies was further analyzed using a set of directlyFITC-labeled antibodies. As before, labeled antibodies were incubatedwith HepG2 cells at 4° C. allowing the antibodies to bind to ASGPR H1but not to internalize. After 45 min incubation with the antibodies (30μg/ml) on a shaker at 4° C., unbound antibodies were removed by washingtwice with cold PBS and cells were re-suspended and cultured inpre-warmed medium at 37° C. to re-activate the cellular metabolismincluding receptor-mediated endocytosis. A cell aliquot was taken after0, 5, 15, 30 and 120 min and washed with cold PBS to stop furtherinternalization. Cell surface-bound antibodies were detected usingPE-conjugated AffiniPure goat anti-human IgG Fc gamma-specific secondaryF(ab′)2 antibody Fragment (Jackson ImmunoResearch #109-116-170, workingsolution 1:50). As seen before, the detection level of surface-exposeddecreased significantly during the first 30 min before it stabilized(FIG. 7C). However, detection of the IgGs by FITC signal, representingboth surface-exposed and internalized antibodies, revealed that thetotal amount of antibody stayed constant over time (FIG. 7D). Thisresult strongly supports the finding that antibodies are in a dynamicsteady state condition of constant internalization and recycling.

Generation of a Clone 51A12-Based L3 Affinity Library

Analysis of the antibody sequences revealed two hot spots in the CDR3region of the 51A12 light chain, namely two adjacent cysteines and aglycosylation site (FIG. 8). For the generation of 51A12-derived cloneswithout cysteines and glycosylation, a maturation library randomized inLCDR3 was generated. The sequence of clone 51A12 (A82G, C112S, C113S,S116A) (SEQ ID NO: 33) was used as a template for the randomization.Triplets encoding positions “RDISSNRAVRN” were randomized throughout thesegment. For the generation of the library, a DNA portion resulting froma two-fragment overlap PCR product was cloned into the phage vector. Forthe generation of fragment 1, the primer combination LCDR3 rand (SEQ IDNO: 151) and fdseqlong (SEQ ID NO: 147) (Table 1 and 3) were used, usingclone 51A12 (A82G, C112S, C113S, S116A) as a template. Amplificationconditions included an initial 5 min 94° C. incubation step followed by25 cycles, each consisting of a 30 sec 94° C. denaturation, a 30 sec 60°C. annealing, and a 90 sec 72° C. elongation step, followed by a final10 min 72° C. elongation step. The resulting fragment was purified on anagarose gel. Fragment 2 was generated with the primer combinationLCDR3rev (SEQ ID NO: 150) and LMB3 (SEQ ID NO: 146) (Table 1 and 3).Amplification conditions included an initial 5 min 94° C. incubationstep followed by 25 cycles, each consisting of a 30 sec 94° C.denaturation, a 30 sec 60° C. annealing, and a 30 sec 72° C. elongationstep, followed by a final 10 min 72° C. elongation step. For theassembly of both fragments, equimolar amounts of fragment 1 and 2 wereused. Amplification conditions included an initial 5 min 94° C.incubation step followed by 5 cycles without primers, each cycleconsisting of a 1 min 94° C. denaturation, a 1 min 60° C. annealing, anda 120 sec 72° C. elongation step. After the addition of the outerprimers LMB3 and fdseqlong, 20 additional cycles were performed usingthe same parameters. At the end, a final 10 min 72° C. incubation stepwas performed. Both, the resulting gel-purified DNA fragment and clone51A12 (A82G, C112S, C113S, S116A) (SEQ ID NO: 33) were digested withNcoI/PstI (FIG. 9). For generation of the library, ligation wasperformed with 10 μg insert and 30 μg vector. Purified ligation wastransformed into TG1 bacteria by electroporation resulting in 3×10⁹transformants. Phagemid particles displaying the Fab library wererescued and purified by PEG/NaCl purification to be used for selections.

TABLE 3 Sequences of primers used for the generation ofthe L3 affinity maturation library. SEQ ID NAME SEQUENCE 150 LCDR3revGGAGTTACAGTAATAGTCAGCCTC 151 LCDR3 randGAGGCTGACTATTACTGTAACTCC 1-2-3-4-5-6-7-8-9-10-11TTCGGCGGAGGGACCAAGCTGACCGTC1: 50% R, 3.1% Rest (no S, T, C); 2: 50% D, 2.8% Rest (no C); 3:50% I, 2.8% Rest (no C); 4: 50% S, 2.8% Rest (no C); 5: 50% S,2.8% Rest (no C); 6: 50% N, 2.8% Rest (no C); 7: 50% R, 2.8% Rest(no C); 8: 50% A, 3.1% Rest (no S, T, C); 9: 50% V, 2.8% Rest (noC); 10: 50% R, 2.8% Rest (no C); 11: 50% N, 2.8% Rest (no C)

Selection of Affinity Matured 51A12-Derived Clones without Cysteines andGlycosylation Site

Generation of affinity-matured 51A12-derived Fabs without cysteines andglycosylation site within LCDR3 was carried out by phage display usingstandard protocols (Silacci et al. (2005), Proteomics 5, 2340-50). Inthe first panning round, selection was carried out in solution accordingto the following procedure: (1) binding of −10¹² phagemid particles to10 nM biotinylated Fc-CRD for 0.5 h in a total volume of 1 ml, (2)capture of biotinylated Fc-CRD and specifically bound phage particles byaddition of 5.4×10⁷ streptavidin-coated magnetic beads for 10 min, (3)washing of beads using 5×1 ml PBS/Tween-20 and 5×1 ml PBS, (4) elutionof phage particles by addition of 1 ml 100 mM TEA for 10 min andneutralization by adding 500 μl 1M Tris/HCl pH 7.4, (5) re-infection ofexponentially growing E. coli TG1 bacteria, and (6) infection withhelperphage VCSM13 and subsequent PEG/NaCl precipitation of phagemidparticles to be used in subsequent selection rounds. Selections werecarried out over three rounds using decreasing (from 10×10⁻⁹M to0.5×10⁻⁹M) antigen concentrations. In round 2 and 3, capture ofantigen-phage complexes was performed using neutravidin plates insteadof streptavidin beads. In addition, neutravidin plates were washed for 3h in 2 l PBS. Specific binders were identified by ELISA as follows: 100μl of 50 nM biotinylated Fc-CRD per well were coated on neutravidinplates. Fab-containing bacterial supernatants were added and bindingFabs were detected via their Flag-tags by using an anti-Flag/HRPsecondary antibody. ELISA-positive clones were bacterially expressed assoluble Fab fragments in 96-well format and supernatants were subjectedto a kinetic screening experiment by SPR-analysis using Proteon XPR36.Clones expressing Fabs with the highest affinity constants wereidentified and the light chains of the corresponding phagemids weresequenced (51A12_C1, SEQ ID NO: 35; 51A12_C7, SEQ ID NO: 37; 51A12_E7,SEQ ID NO: 39; 51A12_H3, SEQ ID NO: 41; 51A12_A6, SEQ ID NO: 43;51A12_D1, SEQ ID NO: 45; 51A12_H6, SEQ ID NO: 47). All clones weredevoid of any critical amino acids in the CDR3 region of the lightchain.

Affinity Determination of the 51A12-Based Affinity Matured Clones by SPR

Affinity (K_(D)) of purified 51A12-derived Fab fragments consisting ofthe parental heavy chain (SEQ ID NO: 4) and the affinity-matured lightchains (51A12_C1, SEQ ID NO: 36; 51A12_C7, SEQ ID NO: 38; 51A12_E7, SEQID NO: 40; 51A12_H3, SEQ ID NO: 42; 51A12_A6, SEQ ID NO: 44; 51A12_D1,SEQ ID NO: 46; 51A12_H6, SEQ ID NO: 48) was measured by surface plasmonresonance using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated mono-(avi-Fc-human ASGPR H1 CRD, SEQ ID NO: 118) orbivalent (avi-Fc-human ASGPR H1 stalk-CRD, SEQ ID NO: 130) ASGPR H1antigens immobilized on NLC chips by neutravidin capture. Immobilizationof recombinant antigens (ligand): Antigens were diluted with PBST (10 mMphosphate, 150 mM NaCl pH 7.4, 0.005% Tween-20) to 10 μg/ml, theninjected at 30 μl/min at varying contact times, to achieveimmobilization levels of 200, 400 or 800 response units (RU) in verticalorientation. Injection of analytes: For one-shot kinetics measurements,injection direction was changed to horizontal orientation, two-folddilution series of purified Fab (varying concentration ranges between12.5 and 0.78 nM) were injected simultaneously at 100 μl/min alongseparate channels 1-5, with association times of 150 or 200 s, anddissociation times of 3600 s. Buffer (PBST) was injected along the sixthchannel to provide an “in-line” blank for referencing. Association rateconstants (k_(on)) and dissociation rate constants (k_(off)) werecalculated using a simple one-to-one Langmuir binding model in ProteOnManager v3.1 software by simultaneously fitting the association anddissociation sensorgrams. The equilibrium dissociation constant (K_(D))was calculated as the ratio k_(off)/k_(on). Regeneration was performedin horizontal orientation using 10 mM glycine, pH 1.5 at a flow rate of100 μl/min for a contact time of 30 s. While most of the selected clonesshowed similar affinities like the parental clone, clone 51A12_A6 (SEQID NO: 44) showed a significantly improved affinity (Table 4).

TABLE 4 Kinetic and thermodynamic parameters of affinity-maturedanti-ASGPR1 Fabs. ASGPR CRD-specific human/cyno ASGPR1 CRD binderska(1/Ms) kd(1/s) KD(M) 51A12 1.10E+05 6.28E−05 5.70E−10 51A12 A82G S116A1.27E+05 1.60E−04 1.25E−09 51A12 S116A 1.31E+05 1.78E−4  1.43E−0951A12_C1 1.74E+05 4.19E−05 2.41E−10 51A12_E7 2.15E+05 9.64E−05 4.48E−1051A12_H3 1.63E+05 8.30E−05 5.10E−10 51A12_A6 3.26E+05 2.61E−05 8.01E−1151A12_C7 1.99E+05 4.67E−05 2.35E−10 51A12_D1 4.00E+05 8.85E−05 2.21E−1051A12_H6 0.86E+05 2.79E−05 3.25E−10

Binding Analysis of the Affinity Matured 51A12 Derivatives to HepG2Cells

Binding of the selected affinity matured 51A12 derivatives to theASGPR-positive hepatocellular carcinoma cell line HepG2 was measured byFACS. As a negative control, the ASGPR-negative cell line Hela was used.0.2 mio cells per well in a 96 well round bottom plate were incubated in300 μl with either purified Fab fragments (1.1, 3.3 and 10 μg/ml) orhuman IgG₁-converted antibodies (0.01, 0.04, 0.1, 0.4, 1.1, 3.3 and 10μg/ml) for 30 min at 4° C. Unbound molecules were removed by washing thecells with PBS containing 0.1% BSA. Bound molecules were detected witheither a FITC-conjugated AffiniPure goat anti-human F(ab′)2fragment-specific secondary F(ab′)2 fragment (Jackson Immuno ResearchLab #109-096-097) or a FITC-conjugated AffiniPure goat anti-human IgG Fcgamma fragment-specific secondary F(ab′)2 fragment (JacksonImmunoResearch #109-096-098; working solution 1:20 in PBS, 0.1% BSA).After 30 min incubation at 4° C. unbound antibody was removed by washingand cells were fixed using 1% PFA. Cells were analyzed using BD FACSCantoll (Software BD DIVA). Analysis of the Fab binding to HepG2 cellsrevealed strong binding of all clones (FIG. 10). Variant 51A12_A6 (SEQID NO: 44) was the strongest binder in both SPR analysis and the cellbinding study. Binding analysis of the clone variants as IgG₁-convertedantibodies to HepG2 cells resulted in a similarly strong binding patternfor all clones (FIG. 11A) while binding to Hela cells at the highestantibody concentration was very weak or not detectable (FIG. 11B),underlining the specificity of these clone variants.

Generation of IgG-IFNα DNA Constructs

DNA sequences encoding ASGPR H1-targeted IgG-IFNα fusion proteins weregenerated based on the ASGPR H1-antibodies 51A12, 51A12 (S116A), 51A12(A82G, S116A), 52C4, 5A4, 4F3, R5C2, R9E10, R7E12, 51A12_C1, 51A12_C7,51A12_E7, 51A12_H3, 51A12_A6, 51A12_D1 and 51A12_H6 wherein oneInterferon-α2a (IFNα) was fused to the C-terminus of one heterodimericheavy chain as shown in FIG. 12A. Targeting to the liver hepatocyteswhere ASGPR H1 is selectively expressed is achieved via the bivalentantibody Fab region (avidity effect). Heterodimerization resulting inthe presence of a single IFNα is achieved by application of theknob-into-hole (kih) technology. In order to minimize the generation ofhomodimeric IgG-cytokine fusions, the cytokine was fused to theC-terminus (with deletion of the C-terminal Lys residue) of theknob-containing IgG heavy chain via a (G₄5)₃ linker. Theantibody-cytokine fusion has IgG-like properties. To reduce FcγRbinding/effector function and prevent FcR co-activation, P329G L234AL235A (LALA) mutations were introduced in the Fc domain. However, FcRnbinding is not impaired. The DNA sequences encoding theseimmunoconjugates are given in SEQ ID NOs 49, 51 and 53 (51A12), SEQ IDNOs 55, 57 and 59 (52C4), SEQ ID NOs 93, 51 and 53 (51A12_A82G, S116A),SEQ ID NOs 91, 51 and 53 (51A12, S116A), SEQ ID NOs 61, 63 and 65 (5A4),SEQ ID NOs 67, 69 and 71 (4F3), SEQ ID NOs 73, 75 and 77 (R5C2), SEQ IDNOs 79, 81 and 83 (R9E19), SEQ ID NOs 85, 87 and 89 (R7E12), SEQ ID NOs95, 51 and 53 (51A12_C1), SEQ ID NOs 97, 51 and 53 (51A12_C7), SEQ IDNOs 99, 51 and 53 (51A12_E7), SEQ ID NOs 101, 51 and 53 (51A12_H3), SEQID NOs 103, 51 and 53 (51A12_A6), SEQ ID NOs 105, 51 and 53 (51A12_D1),SEQ ID NOs 107, 51 and 53 (51A12_H6). In addition, an alternativehole-heavy chain was created where both VH and CH1 domains were deleted(SEQ ID NO: 115). The resulting Fc fragment was able to hetero-dimerizewith the full-length knob heavy chain leading to a monovalent antibodywith a single cytokine fusion (FIG. 12B). As a negative control forfunctional assays, corresponding DNA constructs encoding a controlDP47GS/DPL16 non-targeted IgG-IFNα protein wherein the IgG does not bindto a specified target was generated. The DNA sequence of this isotypeimmunoconjugate is given in SEQ ID NOs 109, 111 and 113.

Expression and Purification of the Antibody-Cytokine Constructs

Immunoconjugates were produced by co-transfecting exponentially growingHEK293-EBNA cells with the mammalian expression vectors using calciumphosphate-transfection. Alternatively, HEK293-EBNA cells growing insuspension were transfected by polyethylenimine (PEI) with therespective expression vectors. Subsequently, the IgG-cytokine fusionproteins were purified from the supernatant by a method composed of oneaffinity step (protein A) followed by size exclusion chromatography(Superdex 200, GE Healthcare). The protein A column (HiTrap ProtA, GEHealthcare) was equilibrated in 20 mM sodium phosphate, 20 mM sodiumcitrate pH 7.5. After loading of the supernatant, the column was firstwashed with 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5 andsubsequently washed with 13.3 mM sodium phosphate, 20 mM sodium citrate,500 mM sodium chloride, pH 5.45. The IgG-cytokine fusion protein waseluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mMglycine, pH 3. Fractions were neutralized, pooled, and purified by sizeexclusion chromatography (HiLoad 16/60 Superdex 200, GE Healthcare) infinal formulation buffer (25 mM potassium phosphate, 125 mM sodiumchloride, 100 mM glycine pH 6.7 or 20 mM histidine, 140 mM NaCl, pH6.0). The protein concentration of purified protein samples wasdetermined by measuring the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence. Purity and molecular weight of immunoconjugates were analyzedby SDS-PAGE or Caliper in the presence and absence of a reducing agent(5 mM 1,4-dithiotreitol). The NuPAGE® Pre-Cast gel system (Invitrogen)was used according to the manufacturer's instructions (4-20%Tris-glycine gels or 3-12% Bis-Tris). The aggregate content ofimmunoconjugate samples was analyzed using a Superdex 200 10/300GLanalytical size-exclusion column (GE Healthcare) in 2 mM MOPS, 150 mMNaCl, 0.02% NaN₃, pH 7.3 running buffer at 25° C. A summary of theanalytical data is shown for selected clones in FIG. 13 (51A12 kih IgGIFNα, SEQ ID NOs 50, 52, 54), FIG. 14 (4F3 kih IgG IFNα, SEQ ID NOs 68,70, 72), FIG. 15 (51A12_C1 kih IgG IFNα, SEQ ID NO: 96, 52, 54), FIG. 16(51A12_E7 kih IgG IFNα, SEQ ID NO: 100, 52, 54), FIG. 17 (51A12_C7 kihIgG IFNα, SEQ ID NO: 98, 52, 54), FIG. 18 (untargeted kih IgG IFNα, SEQID NO: 110, 112, 114) and FIG. 19 (monovalent 51A12 kih IgG IFNα, SEQ IDNO: 50, 52, 116).

Affinity-Determination of IgG-IFNα Immunoconjugates to ASGPR H1 by SPR

The ASGPR H1 binding activity of clones 51A12 and 52C4 used as exemplaryIgG-IFNα immunoconjugates was determined and compared to thecorresponding unmodified IgG antibodies by surface plasmon resonance(SPR) on a ProteOn XPR36 instrument (Biorad). Biotinylated avi-Fc humanASGPR H1 CRD antigen was immobilized on NLC chips by neutravidincapture. Immobilization of recombinant antigens (ligand): Antigens werediluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4,0.005% Tween-20) to 10 μg/ml, then injected at 30 μl/min at varyingcontact times, to achieve immobilization levels of 400 response units(RU) in vertical orientation. Injection of analytes: For one-shotkinetics measurements, injection direction was changed to horizontalorientation, two-fold dilution series of purified IgGs, mono- andbivalent antibody-cytokine fusions (varying concentration ranges between50 and 3.25 nM) were injected simultaneously at 50 μl/min along separatechannels 1-5, with association times of 120 or 200 s, and dissociationtimes of 300 s. Buffer (PBST) was injected along the sixth channel toprovide an “in-line” blank for referencing. Association rate constants(k_(on)) and dissociation rate constants (k_(off)) were calculated usinga simple one-to-one Langmuir binding model in ProteOn Manager v3.1software by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) wascalculated as the ratio k_(off)/k_(on). Regeneration was performed inhorizontal orientation using 10 mM glycine, pH 1.5 at a flow rate of 100μl/min for a contact time of 30 s. The data show that—within the errorof the method—the affinity (monovalent display) and avidity (dimericdisplay) for human ASGPR H1 is retained for both clone 51A12-based (SEQID NOs 50, 52, 54) and clone 52C4-based (SEQ ID NOs 56, 58, 60)immunoconjugate (Table 5).

TABLE 5 Kinetic and thermodynamic parameters of the monovalent andbivalent binding formats of clone 51A12 and 52C4 to ASGPR H1. # ofbinding human/cyno ASGPR1 CRD Name of the binder arms k_(on) (1/Ms)k_(off) (1/s) K_(D)(M) 51A12 IgG 2  3.5 × 10⁵ 7.34 × 10⁻⁵  2.1 × 10⁻¹⁰51A12 kih IgG-IFNα 2 6.74 × 10⁵ 15.7 × 10⁻⁵ 2.33 × 10⁻¹⁰ 51A12 Fab 11.10 × 10⁵ 6.28 × 10⁻⁵ 5.71 × 10⁻¹⁰ monovalent 51A12 1 2.45 × 10⁵ 13.9 ×10⁻⁵ 5.68 × 10⁻¹⁰ kih IgG-IFNα 52C4 IgG 2 5.57 × 10⁵ 49.9 × 10⁻⁵ 0.89 ×10⁻⁹  52C4 kih IgG-IFNα 2 4.26 × 10⁵ 49E×10⁻⁵ 1.15 × 10⁻⁹  52C4 Fab 11.39 × 10⁵ 396 × 10⁻⁵ 28.6 × 10⁻⁹  monovalent 52C4 1 1.14 × 10⁵ 302 ×10⁻⁵ 26.5 × 10⁻⁹  kih IgG-IFNα

Binding of IgG-IFNα Immunoconjugates to ASGPR-Positive and -NegativeCells

In order to characterize the specificity of the antibody conjugates,antibody-cytokine conjugates were incubated with both ASGPR-positive andnegative cells and specific binding was measured by FACS analysis. Forthis, primary human hepatocytes (from 3 donors; purchased from Celsis InVitro Technologies (Baltimore, Md.)), Huh-7 cells, HepG2 cells, A549cells, Hela cells, and 293T cells (each 1×10⁵) were incubated with 1 μgof ASGPR H1-specific IgG kih IFNα samples for 45 min on ice. Afterwashing, the cells were incubated with a labeled goat anti-human IgGsecondary antibody (BD Biosciences, San Diego, Calif.) for 30 min onice. After three washes, the stained cells were analyzed by FACSanalysis using a Calibur flow cytometer. In all FACS assays, an isotypecontrol conjugate (untargeted kih IgG IFNα, SEQ ID NOs 110, 112, 114)was used to determine the background, which was subtracted from the MFIvalues for the tested antibodies. Binding analysis to human peripheralblood mononuclear cells (PBMC) was performed by using directly labeledantibody conjugates (Zenon® R-Phycoerythrin Human IgG Labeling Kit, LifeTechnologies) according to manufacturer's instructions. Binding analysisrevealed that clone 51A12 IgG kih IFNα (SEQ ID NOs 50, 52, 54) and 4F3IgG kih IFNα (SEQ ID NOs 68, 70, 72) showed highly specific binding toASGPR-positive cells while the signal on ASGPR-negative cells wascomparable to the isotype control conjugate (FIG. 20). In addition,binding saturation curves of clone 4F3 IgG kih IFNα were analyzed. Forthis, the antibody-IFNα conjugate was incubated with primary humanhepatocytes (from 3 donors) in a dilution row ranging from 0.0001 to 6.7μg/ml and binding intensity was recorded by FACS analysis. As shown inFIG. 21 binding saturation on primary human hepatocytes as well as onthe control cell line HepG2 was reached at 0.25-0.74 μg/ml antibodyconcentrations, and higher antibody concentration did not significantlyincrease the binding signal further.

Analysis of the Surface-Exposed ASGPR Level on HepG2 Cells Over Time

Uptake of desialylated glycoproteins into liver cells after binding toASGPR is known to occur very rapidly. During this receptor-mediatedendocytosis, the lumen of the endosome becomes acidic allowing thereceptor-ligand complexes to dissociate. While the ligand is targetedfor degradation in lysosomes, ASGPR was shown to recycle back to thecell surface. Since receptor binding followed by internalization wasshown for several receptors to trigger down-regulation of receptorexpression, the levels of surface-exposed ASGPR in the presence of antiASGPR H1 antibodies was measured over time. For this experiment, HepG2cells were incubated for up to 5 h with clone 51A12-derived antibodies,either as IgG or as mono- or bivalent antibody-IFNα fusion proteins. Asa negative control, an unrelated antibody without binding specificity toHepG2 cells was used (GA101) (all antibodies at 30 μg/ml). Duringincubation at 37° C., samples were taken after 30, 60, 120, 180, and 300min and washed with cold PBS. Cell surface bound antibodies weredetected using an APC-conjugated goat anti-human IgG Fcg fragmentspecific F(ab′)2 fragment (Jackson Immuno Research Lab, working solution1:50). After 30 min incubation at 4° C. unbound antibody was removed bywashing with PBS containing 0.1% BSA. Cells were fixed using 1% PFA andanalyzed using BD FACS Cantoll (Software BD DIVA). In order to verifythe integrity of the antibody-cytokine fusion, the presence of IFNα wasalso detected. Cells were incubated with a mouse monoclonal antibodyagainst human interferon alpha (MMHA-1, #21105-1, R&D Systems, 5 μg/ml)for 30 min at 4° C. Unbound antibody was removed by washing with PBScontaining 0.1% BSA and a FITC-conjugated anti-mouse F(ab′)2 Fragment(Serotec, STAR105F; working solution 1:50) was used as secondaryantibody. Cells were fixed using 1% PFA and analyzed using BD FACSCantoll (Software BD DIVA). The results shown in FIG. 22 demonstrate theconstant level of surface-exposed antibody bound to ASGPR without anybinding-induced down-regulation of the receptor over the measured timeperiod. Of note, the monomeric IgG-IFNα construct gave the strongestsignal, most likely due to the fact that twice the number of monomericIgG-IFNα molecules can bind per ASGPR complex (FIG. 22A).

Confocal Microscopy

Three-dimensional and time-resolved analysis of the ASGPR-mediatedinternalization of the antibody-cytokine fusion constructs was performedby confocal microscopy. For this analysis, HepG2 cells were grown to50-60% confluency on glass-bottom dishes (Nunc) in a cell incubator. Thedishes were then rinsed twice with pre-warmed PBS (37° C.) to replacethe medium with PBS and quickly placed on the microscope stage (at 37°C., 5% CO₂). For this experiment, clone 51A12 kih IgG IFNα was directlylabeled with Alexa488. The labeled construct (20 μg/ml) was added toHepG2 cells directly at the microscope stage. Acquisitions started 5 minafter antibody addition using a spinning-disk confocal microscope. Dataacquisitions occurred every 3 sec for 1 h (100× magnification) on 10stacks (z-level) that covered the entire cell thickness. Binding of theantibody-cytokine construct to surface-exposed ASGPR was not equallydistributed but found to be clustered (FIG. 23A). Clusters were spreadover the whole surface. Time-resolved analysis of this experimentclearly revealed the immediate internalization of the antibody-cytokinefusion construct within minutes (FIG. 23B). After internalization of theIgG-cytokine constructs in vesicles, the proteins are then transportedback to the surface on the apical side of the cell (data not shown).

Affinity-Determination of IgG-IFNα Immune Conjugates to Interferon-AlphaReceptor 2 by SPR

The binding activity of IgG-IFNα immune conjugates to the high affinityInterferon-alpha receptor 2 (IFNAR2) was determined and compared toRoferon by surface plasmon resonance (SPR) on a ProteOn XPR36 instrument(Biorad). Commercially available IFNAR2-Fc fusion proteins (R&D Systems)were immobilized in vertical orientation on the sensorchip surface bystandard amine coupling. For one-shot kinetics measurements, injectiondirection was changed to horizontal orientation, two-fold dilutionseries of purified antibody-cytokine fusions (varying concentrationranges between 50 and 3.25 nM) were injected simultaneously at 50 μl/minalong separate channels 1-5, with association times of 120 or 200 s, anddissociation times of 300 s. Buffer (PBST) was injected along the sixthchannel to provide an “in-line” blank for referencing. Association rateconstants (k_(on)) and dissociation rate constants (k_(off)) werecalculated using a simple one-to-one Langmuir binding model in ProteOnManager v3.1 software by simultaneously fitting the association anddissociation sensorgrams. The equilibrium dissociation constant (K_(D))was calculated as the ratio k_(off)/k_(on). Regeneration was performedin horizontal orientation using 10 mM glycine, pH 1.5 at a flow rate of100 μl/min for a contact time of 30 s. The measured affinity ofantibody-cytokine fusion protein was around (k_(on), 1.57×10⁶ 1/Ms;k_(off) 6.15×10⁻³ 1/S; K_(D) 4 nM) and thus comparable to the publishedaffinity of recombinantly produced protein Roferon, indicating that thefusion of IFNα to the C-terminal end of an IgG has no impact on thebinding affinity to IFNAR2.

Determination of the Antiviral Activity of ASGPR mAb-IFNα

In order to analyze the functional activity of IFNα as part of theIgG-cytokine fusion, and to compare it with Roferon, biological activityof the IFNα fusion constructs was tested in a virus protection assay.For this study, MDBK cells were pre-incubated with either Roferon or theantibody-cytokine fusions for 1-4 h. Vesicular stomatitis virus was thenadded for additional 16-24 h. At the end of this incubation step, livingcells were stained with crystal violet staining solution (0.5%) andquantification of living cells was performed using a microplate readerat 550-600 nm with a reference wavelength of 690 nm. Biological activityof all IgG-cytokine constructs was determined in a full dose-responsecurve analysis against a standardized Roferon solution with a 4Parameter-Logistics fitting function. As shown in Table 6, theantibody-IFNα fusion constructs show an activity that corresponds toabout 5% of Roferon's activity, independent of the antibody's bindingvalency. Since the fusion of IFNα to the C-terminal end of an IgG has noimpact on the binding affinity to IFNAR2 (shown above) it is likely thatthe interaction to the low affinity interferon-alpha receptor 1 (IFNAR1)is sterically impaired, ultimately leading to a reduced signaling of theIFNAR holocomplex.

Antiviral Activity of the ASGPR-Specific IgG-IFNα Conjugates in HCVReplicon and EMCV CPE Assays

In order to characterize and compare the functional activity of theIgG-IFNα fusion proteins with commercially available Roferon and Pegasys(peginterferon-α2a), antiviral activity was studied using ASGPR-positive(Huh7-2209-3) and ASGPR-negative cells (Hela).

In order to analyze the antiviral activity of the compounds onASGPR-negative cells, HeLa cells were seeded at 15,000/well in 96-wellopaque-walled plate. After overnight culture, the wells were evacuatedand 50 μl antibody-cytokine conjugate diluted in EMEM (with 10% FBS) wasadded. The HeLa cells were pre-treated with the IgG-IFNα constructs for3 h at 37° C., before the 50 μl EMCV (VR-1762, ATCC) were added intoeach well (2,000 TCID50/well in EMEM). The viable cells were measured 24h post infection using the CellTiter-Glo kit (G7572, Promega). 100 μlCellTiter-Glo reagent was added to each well and incubated at roomtemperature for 10 min with gentle shaking. Then the luminescence signalwas recorded by using a Berthold Mithras Luminometer (BertholdTechnologies). The results represent percentage of survival cells (FIG.24A) and all EC₅₀ values as well as the number of experimentalreiterations are summarized in Table 7. On ASGPR-negative Hela cells,the EC50 values for Roferon are up to 75-fold smaller than for othercompounds such as 4F3 IgG kih IFNα, indicating that the functionalactivity of this compound is much higher than of the other compoundstested. In contrast, the activity of Pegasys was comparable to those ofthe ASGPR-specific IgG kih IFNα conjugates.

The ASGPR-positive Huh 7-derived hepatocarcinoma cell line 2209-23 wasdeveloped by stable transfection of a bicistronic HCV replicon of whichthe first open reading frame, driven by the HCV IRES, contains therenilla luciferase gene in fusion with the neomycin phosphotransferasegene (NPTII) and the second open reading frame, driven by EMCV IRES,contains the HCV non-structural genes NS3, NS4a, NS4b, NS5A and NS5Bderived from the NK5.1 replicon backbone. Cells were cultured at 37° C.in a humidified atmosphere with 5% CO₂ in DMEM supplemented withGlutamax™ and 100 mg/ml sodium pyruvate (#10569-010). The medium wasfurther supplemented with 10% (v/v) FBS (#10082-139), 1% (v/v)penicillin/streptomycin (#15140-122) and 1% (v/v) geneticin. Allreagents were obtained from Invitrogen.

Huh 7 2209-23 cells in DMEM containing 5% (v/v) Fetal Bovine Serum wereplated in 96-well plates at 5000 cells/well in 90 μl volume. 24 hoursafter plating, antibody-cytokine conjugates (or medium as a control)were added to the cells in 3-fold dilutions over 12 wells (0.01-2000pM), in a volume of 10 μl. Final volume after addition of compound was100 μl. Renilla luciferase reporter signal was read 72 hours afteradding compounds, using the Renilla Luciferase Assay system (Promega, #E2820). The EC₅₀ values were calculated as the compound concentration atwhich a 50% reduction in the level of renilla luciferase reporter wasobserved as compared to control samples (in the absence of compound).Dose-response curves and EC₅₀ values were obtained by using the XLfit4program (ID Business Solutions Ltd., Surrey, UK). Despite the reducedantiviral activity of the antibody-cytokine constructs when exposed toASGPR-negative cells (Table 6 and FIG. 24A), clones 51A12 IgG kih IFNαand 4F3 IgG kih IFNα were more potent in protecting cells from viralinfection and multiplication than Roferon when incubated with theASGPR-positive cell line Huh? 2209-23 (FIG. 24B and Table 7). Incontrast, the potency of the isotype control (untargeted IgG kih IFNα)was significantly lower, underlining the positive consequence oftargeting the IgG kih IFNα conjugates to ASGPR H1.

TABLE 7 Summary of antiviral activity of various ASGPR H1-specificantibody-IFNα conjugates. HCV Replicon (Huh-7) EMCV assay (Hela) IFNaMean EC50 Mean EC50 Molecule (pM) STDEV n = (pM) STDEV n = Roferon 0.070.03 4 1.8 0.4 6 Pegasys 0.96 0.46 5 61.2 9.4 9 Isotype-IFN 0.59 0.12 3108.3 5.0 6 51A12-IFN 0.02 0.01 4 68.2 14.4 6 4F3-IFNa 0.04 0.01 4 135.629.1 6 R7E12-IFN 0.11 0.04 3 29.2 2.3 6 R9E10-IFN 0.43 0.21 3 49.0 5.6 6R5C2-IFN 0.54 0.23 3 56.3 4.9 6 5A4-IFN 0.86 0.4 3 58.7 5.8 6

IFNα Activity of the ASGPR-Specific IgG-IFNα Conjugates in Hepatic andNon-Hepatic Cells

IFNα exerts its antiviral activities through induction of hundreds ofIFN-stimulated genes (ISG). To verify the antiviral activities, andfurther confirm the ASGPR-targeting mediated enhanced IFNα activity, wedetermined the ISG expression in hepatic and non-hepatic cells. Hepaticcells (primary hepatocytes and HepG2) and non-hepatic cells (human PBMCand Hela) were treated with various serially diluted IFNα molecules for6 h and total RNA was extracted from cells using 5PRIME RNA extractionkit (#FP2302530, 5PRIME, Gaithersburg, Md.).

TaqMan (real-time PCR) assays for ISG genes MX1 and RSAD2 were customdesigned. Assays were selected to lie within the Affymetrix probesequence of interest or within the 3′ coding sequence of the referencemRNAs of interest.

All gene expression assays were performed on an ABI PRISM® 7900HTSequence Detection System (Applied Biosystems). PCR mix consisted of 10μl PerfeCTa® qPCR FastMix, ROX™ (Quanta), 1 μl TaqMan or 0.06 μl IDTassay, and 2 μl DEPC-treated water (Ambion, Applied Biosystems) for eachreaction. cDNA samples were diluted to 10 ng/μl in RNase-free water(Ambion, Applied Biosystems), and 7 μl added to a 384-well optical plate(Applied Biosystems) containing 13 μl pre-distributed assay PCR mix. Allsamples were queried with one assay for a target gene or one for theendogenous control gene assays, 18S, GAPDH (Rhesus), ACTB (Rhesus) andGUSB (Rhesus) (TaqMan Gene Expression Assays, Applied Biosystems). Eachmeasurement was performed in triplicate. The following PCR conditionswere used: 45° C. for 2 min, then 95° C. for 3 min, followed by 40cycles at 95° C. for 15 s and 60° C. for 45 s.

The expression levels of target genes were normalized to the geometricmean of 18S, ACTB, GAPDH and GUSB and represented as relative expression(E), E=2^((ΔCt)), where ΔCt is the difference between reference andtarget gene cycles at which the amplification exceeds an arbitrarythreshold. As shown in FIG. 25, isotype IgG kih IFNα control showedreduced ISG induction compared to Roferon, with similar activity toPegasys (PEG-IFNα) in all cells. In the ASGPR-negative Hela and PBMCcells, clone 51A12 IgG kih IFNα also showed reduced activity, similar tothe isotype control. However, in the ASGPR-positive hepatic cells HepG2and primary human hepatocytes, clone 51A12 IgG kih IFNα showed enhancedIFNα activity compared to the isotype control, to a similar level ofRoferon. This result confirms the above-described enhanced antiviralactivity of the 51A12 antibody-IFNα conjugate.

In order to understand whether these ASGPR-targeted IFNα molecules havesustained IFNα activity, we monitored ISG expression in Huh-7 andprimary hepatocytes for up to 72 h. As shown in FIG. 26, bothASGPR-targeting IFNα molecules 51A12 IgG kih IFNα and 4F3 IgG kih IFNαshowed sustained ISG induction at 72 h after treatment, while Pegasysand Roferon showed significantly reduced ISG induction at 72 h.

Cynomolgus Monkey Single Dose PK/PD Study

Encouraged by in vitro results that ASGPR antibody-based targeted IFNαmolecules showed reduced IFNα activity in non-hepatic cells and enhancedIFNα activity in hepatic cells (liver-targeted IFNα effects), acynomolgus monkey study was designed to confirm the liver-targeted IFNαeffects in vivo. Because the ASGPR-specific clone 51A12 binds to humanand monkey ASGPR with identical affinity and human IFNα has similaractivity in the monkeys, monkeys can be used as PK/PD models forliver-targeted IFNα proof-of-concept studies. In the monkey study wedirectly compared 51A12 IgG kih IFNα and isotype IgG kih IFNα control.Both molecules were injected subcutaneously at dosage levels of either 1or 10 μg/kg, monkey blood and liver biopsy samples were collected beforeand after dosing, and their PK (pharmacokinetics) and PD(pharmacodynamics) were monitored. The dose groups are listed in Table8.

TABLE 8 Twelve cynomolgus monkeys were divided into four groups as shownbelow. Formulation No of Strength Group Dose (μg/kg), compound Animals(μg/ml) 1 10 μg/kg, 51A12, SC, Single Dose 3 20 2  1 μg/kg, 51A12, SC,Single Dose 3 2 3 10 μg/kg, iso-control, SC, Single Dose 3 20 4  1μg/kg, iso-control, SC, Single Dose 3 2

Sample Collection, Transfer and Storage

Blood (approximately 1 ml) was collected from each animal 5 days beforedosage and at 2, 6, 12, 24, 48, 72, 96, 168, and 336 hours afterinjection. Samples for pharmacokinetics were collected into tubeswithout anticoagulant. Blood for pharmacokinetics was collected prior tothe pharmacodynamic blood collections.

For the gene expression study, blood (approximately 2.5 ml) wascollected from each animal 5 days before dosage and at 2, 6, 12, 24, 48,72, 96, 168, and 336 hours after injection. Samples were collected intoPAXgene™ blood RNA collection tubes and the tubes were mixed byinverting 8-10 times. The PAXgene™ blood RNA collection tube was thelast tube drawn in the phlebotomy sequence (i.e. after the clinicalpathology and pharmacokinetic collections).

Liver tissue was collected from two separate locations (at least 25mg/sample) in the liver via laparoscopic procedure from each animal onday −5, and on day 2, day 4 and day 8. Each tissue sample was excisedand immediately placed into separate pre-weighed and labeled cryo-vialtubes and flash-frozen in liquid nitrogen. Because of the need forimmediate freezing, the sample vials were not weighed after collectionof the liver samples. For day −5 and day 2, the first liver tissuealiquot for each animal was taken from the left lateral lobe of theliver, and the second liver tissue aliquot for each animal was takenfrom the right lateral lobe of the liver. For day 4 and day 8, the firstliver tissue aliquot for each animal was taken from the left medial lobeof the liver, and the second liver tissue aliquot for each animal wastaken from the right medial lobe of the liver. Liver biopsy samples weresnap-frozen in 2 ml tubes. RNAlater-ICE (P/N 7031, Ambion), pre-chilledon dry ice, was added to frozen tissues and stored at −80° C. Bloodsamples were received in PAXgene tubes according to the protocol andstored at −80° C. prior to processing.

Measurement of Clone 51A12 IgG Kih IFNα and Isotype IgG Kih IFNα Controlin Monkey Serum Samples

Aliquots of cynomolgus monkey serum were analyzed for the dosed compoundusing a sandwich ELISA assay that uses an anti-IFNα antibody (Lot no.34495-28, Roche Nutley, N.J., USA) as the capture reagent andHRP-labeled anti-human Fc antibody (Lot no. Wbr72_MM_(—)090602, RocheDiagnostics, Penzberg, Germany) as the detection reagent. After coatingof plates with anti-IFNα antibody at room temperature for 1 h, theplates were treated with 2% BSA blocking buffer for 1 h. After washing,HRP-labeled anti-human Fc antibody was added to each well and incubatedfor 1 h with gentle shaking. After washing, 100 μl/well TMB substratesolution (#11 484 281 001, Roche Diagnostics, Penzberg, Germany) wasadded for about 20 min. The reaction was then stopped by adding 50μl/well 2N HCl. The plates were read within 2 min at 450 nm withreference wavelength of 650 nm. The lower limit of quantitation (LLOQ)of this method was 10 ng/ml. The precision (% CV) and accuracy (%relative error) of the assay met the acceptance criteria. Assayperformance, as monitored by the analysis of QC samples analyzed alongwith the samples, was as shown in Table 9. The serum concentrations areshown in Table 10-13. A single injection of isotype IgG kih IFNα at 10μg/kg yielded significant exposure in the blood that peaked at around100 ng/ml for one week. In contrast, at the same dose level, 51A12 IgGkih IFNα was below quantification level at any time point. Bothmolecules were undetectable in the blood at 1 μg/kg dose level. The PKparameters are summarized in Table 14.

TABLE 9 Analytical performance of clone 51A12 IgG kih IFNα qualitycontrol samples in cynomolgus monkey serum. Run Curve QC1 QC2 QC3 DateNumber 30.0 ng/mL 90.0 ng/mL 270 ng/mL 13 Dec. 2011 1 24.3 78.8 252 29.195.2 236 13 Dec. 2011 2 * 76.4 251 26.5 94.7 247 20 Dec. 2011 3 21.774.7 245 28.6 84.3 264 4 25.4 82.6 288 28.2 91.2 279 Mean 26.3 84.7 258% CV 10.2 9.6 6.9 % Rel. Error −12.3 −5.9 −4.4 * Deactivated

TABLE 10 Serum concentration (ng/ml) of 1 μg/kg isotype IgG kih IFNα.Time % [h] Subject 1 Subject 2 Subject 3 Mean S.D. CV n 0 BLQ <10.0 BLQ<10.0 BLQ <10.0 2 BLQ <10.0 BLQ <10.0 BLQ <10.0 6 BLQ <10.0 BLQ <10.0BLQ <10.0 12 BLQ <10.0 BLQ <10.0 BLQ <10.0 24 BLQ <10.0 BLQ <10.0 BLQ<10.0 48 BLQ <10.0 BLQ <10.0 BLQ <10.0 72 BLQ <10.0 BLQ <10.0 BLQ <10.096 BLQ <10.0 BLQ <10.0 BLQ <10.0 168 BLQ <10.0 BLQ <10.0 BLQ <10.0 336BLQ <10.0 BLQ <10.0 BLQ <10.0

TABLE 11 Serum concentration (ng/ml) of 1 μg/kg 51A12 IgG kih IFNα. Time% [h] Subject 1 Subject 2 Subject 3 Mean S.D. CV n 0 BLQ <10.0 BLQ <10.0BLQ <10.0 2 BLQ <10.0 BLQ <10.0 BLQ <10.0 6 BLQ <10.0 BLQ <10.0 BLQ<10.0 12 BLQ <10.0 BLQ <10.0 BLQ <10.0 24 BLQ <10.0 BLQ <10.0 BLQ <10.048 BLQ <10.0 BLQ <10.0 BLQ <10.0 72 BLQ <10.0 BLQ <10.0 BLQ <10.0 96 BLQ<10.0 BLQ <10.0 BLQ <10.0 168 BLQ <10.0 BLQ <10.0 BLQ <10.0 336 BLQ<10.0 BLQ <10.0 BLQ <10.0

TABLE 12 Serum concentration (ng/ml) of 10 μg/kg 51A12 IgG kih IFNα.Time % [h] Subject 1 Subject 2 Subject 3 Mean S.D. CV n 0 BLQ <10.0 BLQ<10.0 BLQ <10.0 2 BLQ <10.0 BLQ <10.0 BLQ <10.0 6 BLQ <10.0 BLQ <10.0BLQ <10.0 12 BLQ <10.0 BLQ <10.0 BLQ <10.0 24 BLQ <10.0 BLQ <10.0 BLQ<10.0 48 BLQ <10.0 BLQ <10.0 BLQ <10.0 72 BLQ <10.0 BLQ <10.0 BLQ <10.096 BLQ <10.0 BLQ <10.0 BLQ <10.0 168 BLQ <10.0 BLQ <10.0 BLQ <10.0 336BLQ <10.0 BLQ <10.0 BLQ <10.0

TABLE 13 Serum concentration (ng/ml) at 10 μg/kg isotype IgG kih IFNα.Time % [h] Subject 1 Subject 2 Subject 3 Mean S.D. CV n 0 BLQ <10.0 BLQBLQ <10.0 <10.0 2 BLQ <10.0 33.7 12.4 23.1 2 6 49.8 40.9 61.5 50.7 10.320.3 3 12 66.4 44.3 67.9 59.5 13.2 22.2 3 24 109 63.1 71.1 81.1 24.530.2 3 48 119 99.3 88.3 102 15.6 15.3 3 72 105 88.2 94.0 95.7 8.53 8.9 396 124 85.0 95.2 101 20.2 20.0 3 168 78.9 56.5 58.3 64.6 12.4 19.2 3 33619.6 11.0 BLQ <10.0 15.3 2

TABLE 14 Cynomolgus monkey serum PK parameters of 10 μg/kg isotype-IFNα.Parameter Units Subject 1 Subject 2 Subject 3 Mean S.D. % CV n originaldose μg/kg 10 10 10 C_(max) ng/ml 124 99 95 106 16 14.8 3 T_(max) hours96 48 96 80 28 34.6 3 AUC ng * hours/ml 25251 18124 13279 18885 602231.9 3 AUC interval hours (0-336) (0-336) (0-168) AUC/dose ng *hours/ml/μg/kg 2525 1812 1328 1888 602 31.9 3 AUC extrap ng * hours/ml27615 19253 21839 22903 4281 18.7 3 % AUC extrap % 8.6 5.9 39 17.9 18.5104 3 AUC extrap/ ng * hours/ml/μg/kg 2762 1925 2184 2290 428 18.7 3dose T_(1/2) hours 83.6 71.1 102 85.5 15.4 18.0 3

RNA Extraction

Total RNA was extracted from 96 liver biopsy samples (48 animals×2biopsies per sampling) using the Qiagen RNeasy Mini Kit (P/N 74104) andquantitated on the Nanodrop 8000. Total RNA quality of the liver sampleswas assessed on the Caliper LabChip GX. RNA from all samples was ofsufficient quantity and quality to perform qPCR and microarray-basedgene expression measurements.

Total RNA isolation and quantitation from 120 blood samples wasperformed at Expression Analysis (Raleigh, N.C.). Total RNA quality ofthe blood samples was assessed on the Agilent Bioanalyzer 2100. RNA fromall samples was of sufficient quantity and quality to perform qPCR andmicroarray-based gene expression measurements.

Microarray Analysis

Two separate protocols were used to convert total RNA into cDNA:Affymetrix GeneChip HT 3′ IVT Express (P/N 901225) and NuGEN Ovation RNAAmplification System V2 (P/N 3100-60).

Blood

100 ng total RNA from liver biopsy samples was converted intodouble-stranded cDNA and amplified RNA (aRNA) using the GeneChip HT 3′IVT Express Kit according to the manufacturer's protocols. Thehybridization mix contained 12.5 μg aRNA, 2× Hybridization Mix (P/N900720), DMSO, 20× Hybridization Controls, and Oligo B2 Controls.

Liver

50 ng total RNA from blood samples was converted to single-stranded cDNAusing the NuGEN Ovation RNA Amplification System V2 kit following themanufacturer's protocol. The hybridization mix contained 3 μgSPIA-amplified cDNA, 2× Hybridization Mix (P/N 900720), DMSO, 20×Hybridization Controls, and Oligo B2 Controls.

The hybridization mixes for liver and blood samples were hybridized toAffymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Staining andwashing steps were performed as suggested by the manufacturer(Affymetrix). Each hybridized Affymetrix GeneChip array was scanned witha GeneChip Scanner 3000 7G (Agilent/Affymetrix). Image analysis wasperformed with the Affymetrix GCOS software. The resulting .cel fileswere assessed using standardized quality control metrics. Data werenormalized and expression value calculation/differential expression wasdetermined using standard data analysis packages.

In FIG. 27 the expression of four representative ISGs HRASLS2, ITI44,IFIT1, and IFITM2 in monkey liver samples is graphed. More robustinduction of the expression of these four ISGs was found in 51A12 IgGkih IFNα dosed monkeys, in comparison to the isotype IgG kih IFNα dosedmonkeys.

IFN Gene Expression Analysis (M3.1 Heatmap)

In order to more comprehensively analyze the IFNα stimulated geneexpression by IFNα molecules, IFNα response was analyzed with IFN genemodules determined from blood transcriptomics studies (Chaussabel et al.(2008), Immunity 29, 150-64). As shown in FIG. 28A, the fold-changeexpression values from baseline for the genes of the interferon moduleM3.1 were plotted in heatmap form for both blood and liver samples usingthe R statistics package (www.r-project.org). Non-supervisedhierarchical clustering of the liver interferon-induced genes reveals ahighly induced subset (dashed rectangle) in the 10 μg/kg dose of 51A12but not the isotype IFNα compound at days 1 and 3. Non-supervisedhierarchical clustering this subset reveals a differential pattern ofexpression between blood and liver where some of the genes were moresignificantly induced in liver by 51A12 but not isotype-IFNα at the 10μg/kg dose and other genes were more significantly induced in blood byisotype IFNα at the high dose (FIG. 28B).

In summary, ASGPR-targeting IFNα molecule 51A12 IgG kih IFNα showedundetectable exposure in the blood, and lower IFNα activity (ISGexpression stimulation) in the blood but higher IFNα activity in monkeyliver, as compared to isotype IFNα control.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. An antibody for specific binding to asialoglycoprotein receptor(ASGPR), wherein the antibody comprises a) a heavy chain variable regionsequence of SEQ ID NO: 16 and a light chain variable region sequence ofSEQ ID NO: 14; b) a heavy chain variable region sequence of SEQ ID NO: 4and a light chain variable region sequence of SEQ ID NO: 2; c) a heavychain variable region sequence of SEQ ID NO: 8 and a light chainvariable region sequence of SEQ ID NO: 6; d) a heavy chain variableregion sequence of SEQ ID NO: 12 and a light chain variable regionsequence of SEQ ID NO: 10; e) a heavy chain variable region sequence ofSEQ ID NO: 20 and a light chain variable region sequence of SEQ ID NO:18; f) a heavy chain variable region sequence of SEQ ID NO: 24 and alight chain variable region sequence of SEQ ID NO: 22; g) a heavy chainvariable region sequence of SEQ ID NO: 28 and a light chain variableregion sequence of SEQ ID NO: 26; h) a heavy chain variable regionsequence of SEQ ID NO: 4 and a light chain variable region sequence ofSEQ ID NO: 30; i) a heavy chain variable region sequence of SEQ ID NO: 4and a light chain variable region sequence of SEQ ID NO: 32; j) a heavychain variable region sequence of SEQ ID NO: 4 and a light chainvariable region sequence of SEQ ID NO: 34; or k) a heavy chain variableregion sequence of SEQ ID NO: 24 and a light chain variable regionsequence of SEQ ID NO:
 22. 2. The antibody of claim 1, wherein theantibody comprises the heavy chain variable region sequence of SEQ IDNO: 16 and the light chain variable region sequence of SEQ ID NO:
 14. 3.An antibody for specific binding to ASGPR, wherein the antibody competeswith the antibody of claim 2 for binding to an epitope of ASGPR. 4-8.(canceled)
 9. The antibody of claim 1, wherein the antibody comprisesthe heavy chain variable region sequence of SEQ ID NO: 4 and the lightchain variable region sequence of SEQ ID NO:
 2. 10. An antibody forspecific binding to ASGPR, wherein the antibody competes with theantibody of claim 9 for binding to an epitope of ASGPR. 11-15.(canceled)
 16. The antibody of claim 10, wherein the antibody comprisesa) a heavy chain variable region sequence of SEQ ID NO: 4 and a lightchain variable region sequence of SEQ ID NO: 36; b) a heavy chainvariable region sequence of SEQ ID NO: 4 and a light chain variableregion sequence of SEQ ID NO: 38; c) a heavy chain variable regionsequence of SEQ ID NO: 8 and a light chain variable region sequence ofSEQ ID NO: 40; d) a heavy chain variable region sequence of SEQ ID NO:12 and a light chain variable region sequence of SEQ ID NO: 42; e) aheavy chain variable region sequence of SEQ ID NO: 20 and a light chainvariable region sequence of SEQ ID NO: 44; or f) a heavy chain variableregion sequence of SEQ ID NO: 24 and a light chain variable regionsequence of SEQ ID NO: 46; or g) a heavy chain variable region sequenceof SEQ ID NO: 28 and a light chain variable region sequence of SEQ IDNO:
 48. 17. The antibody according to claim 1, claim 3, or claim 10,wherein the antibody is capable of specific binding to human andcynomolgus monkey ASGPR. 18-27. (canceled)
 28. The antibody of claim 17,wherein the antibody comprises a human Fc region, selected from thegroup consisting of an IgG Fc region, and an IgG₁ Fc region.
 29. Theantibody of claim 17, wherein the antibody is (i) a full-lengthantibody, (ii) an IgG class antibody, or (iii) an IgG₁ subclassantibody.
 30. (canceled)
 31. The antibody of claim 17, wherein theantibody comprises in the Fc region a modification reducing bindingaffinity of the antibody to an Fc receptor, or an Fcγ receptor; whereinsaid Fc receptor is an activating Fc receptor and wherein the activatingFc receptor is FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), or FcαRI(CD89). 32-33. (canceled)
 34. The antibody of claim 31, wherein said Fcreceptor is FcγRIIIa, or human FcγRIIIa.
 35. The antibody of claim 31,wherein the antibody comprises an amino acid substitution in the Fcregion at a position selected from the group consisting of P329, L234and L235 (EU numbering).
 36. The antibody of claim 31, wherein theantibody comprises the amino acid substitutions P329G, L234A and L235Ain the Fc region (EU numbering). 37-38. (canceled)
 39. The antibody ofclaim 28, wherein the antibody comprises a modification within theinterface between the two antibody heavy chains in the CH3 domain,wherein i) in the CH3 domain of one heavy chain, an amino acid residueis replaced with an amino acid residue having a larger side chainvolume, thereby generating a protuberance (“knob”) within the interfacein the CH3 domain of one heavy chain which is positionable in a cavity(“hole”) within the interface in the CH3 domain of the other heavychain, and ii) in the CH3 domain of the other heavy chain, an amino acidresidue is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity (“hole”) within the interfacein the second CH3 domain within which a protuberance (“knob”) within theinterface in the first CH3 domain is positionable.
 40. The antibody ofclaim 28, wherein the antibody comprises the amino acid substitutionT366W and optionally the amino acid substitution S354C in one of theantibody heavy chains, and the amino acid substitutions T366S, L368A,Y407V and optionally Y349C in the other of the antibody heavy chains.41. The antibody of claim 17, comprising an effector moiety attached tothe antibody, wherein the effector moiety is a cytokine molecule. 42.The antibody of claim 41, wherein no more than one effector moiety isattached to the antibody.
 43. (canceled)
 44. The antibody of claim 41,wherein the cytokine molecule is fused by an amino-terminal amino acidto a carboxy-terminal amino acid of one of the heavy chains of theantibody.
 45. (canceled)
 46. The antibody of claim 41, wherein thecytokine molecule is an interferon molecule selected from the groupconsisting of human interferon alpha, human interferon alpha 2 and humaninterferon alpha 2a. 47-53. (canceled)
 54. A method for producing theantibody of claim 17, comprising the steps of (a) culturing a host cellcomprising (i) a polynucleotide encoding the antibody of claim 17 or anantigen binding portion thereof, or (ii) an expression vector,comprising the polynucleotide of step (i) under conditions suitable forexpression of said antibody, and (b) recovering said antibody. 55.(canceled)
 56. A pharmaceutical composition comprising the antibody ofclaim 17 and a pharmaceutically acceptable carrier. 57-71. (canceled)72. A method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the antibody of claim 17 in a pharmaceuticallyacceptable form.
 73. The method of claim 72, wherein said disease is aliver disease selected from the group consisting of hepatitis virusinfection, HBV infection and the liver cancer hepatocellular carcinoma(HCC). 74-82. (canceled)
 83. A method for targeting a cell expressingASGPR in an individual, comprising administering to said individual acomposition comprising the antibody of claim 17 in a pharmaceuticallyacceptable form, wherein the cell is a hepatocyte. 84-86. (canceled)